Non-Human Animals Comprising SLC30A8 Mutation And Methods Of Use

ABSTRACT

Non-human animal genomes, non-human animal cells, and non-human animals comprising a mutated Slc30a8 locus and methods of making and using such non-human animal genomes, non-human animal cells, and non-human animals are provided. The non-human animals can have increased insulin secretory capacity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 62/584,228,filed Nov. 10, 2017, U.S. Application No. 62/666,337, filed May 3, 2018,and U.S. Application No. 62/689,945, filed Jun. 26, 2018, each of whichis herein incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The Sequence Listing written in file 523054SEQLIST.txt is 39.2kilobytes, was created on Nov. 9, 2018, and is hereby incorporated byreference.

BACKGROUND

Diabetes is a disorder characterized by metabolic defects ininsulin-responsive tissues and insulin-producing pancreatic beta cellsresulting in a failure to maintain appropriate blood sugar levels in thebody. Diabetes in humans can be defined as a disorder corresponding to afasting plasma glucose concentration greater than 125 mg/dL, or a plasmaglucose concentration greater than 199 mg/dL two hours after ingestionof a 75 g oral glucose load. Two major forms of diabetes are type 1diabetes (T1D) and type 2 diabetes (T2D). T1D is an autoimmune disorderresulting in destruction of beta-pancreatic cells and an absolutedeficiency of insulin, the hormone that regulates glucose utilization.By contrast, T2D can occur with normal or even elevated levels ofinsulin from the inability of tissues to respond appropriately toinsulin. Most T2D patients have impaired insulin sensitivity. Insulinsecretion cannot compensate for the resistance of peripheral tissues torespond to insulin. Many T2D patients are obese. Type 1.5 diabetes (lateautoimmune onset in adults) shows some characteristics of T1D and T2D.

Zinc is required for insulin biosynthesis and processing. Two zinc ionsare complexed in a hexameric form of proinsulin, which is ultimatelyprocessed to insulin.

SLC30A8 encodes a zinc transporter that is primarily expressed in thepancreatic islet, where it transports zinc into insulin-containingsecretory granules. Heterozygous loss-of-function (LOF) mutations inSLC30A8 protect from type 2 diabetes in humans. Several Slc30a8 knockoutmouse lines have been characterized, but they do not fully recapitulatethe protective human phenotype. Rather, Slc30a8-deficient mice havereduced plasma insulin levels and impaired glucose tolerance.

SUMMARY

Non-human animals comprising a mutated Slc30a8 locus are provided, aswell as methods of making and using such non-human animals. Non-humananimal genomes or cells comprising a mutated Slc30a8 locus are alsoprovided.

In one aspect, provided are non-human animal genomes, non-human animalcells, or non-human animals comprising a mutated Slc30a8 locus. Suchnon-human animal genomes, non-human animal cells, or non-human animalscan comprise a mutated Slc30a8 gene, wherein the mutated Slc30a8 geneencodes a truncated SLC30A8 protein and results in the non-human animalhaving an enhanced capacity for insulin secretion relative to anon-human animal without the mutation.

In one aspect, provided are non-human animals whose genome comprises anendogenous Slc30a8 locus comprising a mutated Slc30a8 gene, wherein themutated Slc30a8 gene encodes a truncated SLC30A8 protein and results inthe non-human animal having an enhanced capacity for insulin secretionrelative to a non-human animal without the mutation.

In some such non-human animals, the non-human animals exhibit enhancedcapacity for insulin secretion in response to hyperglycemia. Optionally,the non-human animal has increased insulin secretion in response tohyperglycemia induced by insulin receptor inhibition relative to thenon-human animal without the mutation. Optionally, the increased insulinsecretion in response to hyperglycemia induced by insulin receptorinhibition is not associated with increased beta-cell proliferation orbeta-cell mass relative to the non-human animal without the mutation.

In some such non-human animals, the enhanced capacity for insulinsecretion is observed when the non-human animals are fed a high-fatdiet. Optionally, the non-human animal has increased insulin secretionrelative to the non-human animal without the mutation when fed ahigh-fat diet, wherein the increased insulin secretion is associatedwith increased beta-cell proliferation or beta-cell mass relative to thenon-human animal without the mutation. Optionally, the increasedbeta-cell proliferation is insulin-receptor-dependent (e.g., dependenton an insulin receptor in the beta cells).

In some such non-human animals, non-human animal cells, or non-humananimal genomes, the mutated Slc30a8 gene has a premature terminationcodon. In some such non-human animals, non-human animal cells, ornon-human animal genomes, the mutated Slc30a8 gene comprises a mutationis in the third exon of the Slc30a8 gene. Optionally, the mutation is atthe 3′ end of the third exon of the Slc30a8 gene.

In some such non-human animals, non-human animal cells, or non-humananimal genomes, the mutated Slc30a8 gene comprises a nonsense mutation.Optionally, the nonsense mutation is in a codon corresponding to thecodon encoding R138 in SEQ ID NO: 14 when the SLC30A8 protein encoded bythe mutated Slc30a8 gene is optimally aligned with SEQ ID NO: 14.Optionally, the nonsense mutation is at a position corresponding toresidue 412 in SEQ ID NO: 21 when the coding sequence of the mutatedSlc30a8 gene is optimally aligned with SEQ ID NO: 21.

In some such non-human animals, non-human animal cells, or non-humananimal genomes, the mutated Slc30a8 gene is endogenous to the non-humananimal. In some such non-human animals, non-human animal cells, ornon-human animal genomes, the non-human animal is a non-human mammal. Insome such non-human animals, non-human animal cells, or non-human animalgenomes, the non-human animal is a rat or a mouse. Optionally, thenon-human animal is a mouse. Optionally, the mutated Slc30a8 geneencodes a SLC30A8 protein comprising the sequence set forth in SEQ IDNO: 13. Optionally, the mutated Slc30a8 gene comprises the codingsequence set forth in SEQ ID NO: 22 or degenerates thereof that encodethe same amino acid sequence.

Some such non-human animals have increased insulin secretion in responseto hyperglycemia induced by insulin receptor inhibition relative to thenon-human animal without the mutation. In some such non-human animals,the increased insulin secretion is not associated with increasedbeta-cell proliferation or beta-cell mass relative to the non-humananimal without the mutation. Some such non-human animals have decreasedmitochondrial gene expression relative to the non-human animal withoutthe mutation. Some such non-human animals have increased Hvcn1expression relative to the non-human animal without the mutation. Insome such non-human animals, the non-human animal has normal glucosehomeostasis and glucose-induced insulin secretion on a control chow dietrelative to the non-human animal without the mutation. In some suchnon-human animals, the non-human animal has a normal metabolic phenotypeon a control chow diet relative to the non-human animal without themutation.

Some such non-human animals have one or more of the followingcharacteristics relative to the non-human animal without the mutation:(a) increased glucose-induced insulin secretion when fed the high-fatdiet; (b) increased pancreatic beta-cell proliferation when fed thehigh-fat diet; (c) increased number of pancreatic beta cells when fedthe high-fat diet; and (d) increased fed plasma insulin levels afterblockade of the insulin receptor. Optionally, the non-human animal hasall of the following characteristics relative to the non-human animalwithout the mutation: (a) increased glucose-induced insulin secretionwhen fed the high-fat diet; (b) increased pancreatic beta-cellproliferation when fed the high-fat diet; (c) increased number ofpancreatic beta cells when fed the high-fat diet; and (d) increased fedplasma insulin levels after blockade of the insulin receptor.

Some such non-human animals have one or more of the followingcharacteristics relative to the non-human animal without the mutation:(a) increased circulating insulin levels after fed the high-fat diet for20 weeks; (b) increased number of pancreatic beta cells after fed thehigh-fat diet for 20 weeks; (c) decreased proinsulin-to-insulin ratiowhen fed the high-fat diet; and (d) increased fed plasma insulin levelsafter blockade of the insulin receptor. Optionally, the non-human animalhas all of the following characteristics relative to the non-humananimal without the mutation: (a) increased circulating insulin levelsafter fed the high-fat diet for 20 weeks; (b) increased number ofpancreatic beta cells after fed the high-fat diet for 20 weeks; (c)decreased proinsulin-to-insulin ratio when fed the high-fat diet; and(d) increased fed plasma insulin levels after blockade of the insulinreceptor.

In some such non-human animals, Slc30a8 mRNA expression levels in theislets of the non-human animal are at least 25% of Slc30a8 mRNAexpression levels in the islets of the non-human animal without themutation.

Some such non-human animals, non-human animal cells, or non-human animalgenomes are heterozygous for the mutation. Some such non-human animals,non-human animal cells, or non-human animal genomes are homozygous forthe mutation. Some such non-human animals, non-human animal cells, ornon-human animal genomes are male. Some such non-human animals,non-human animal cells, or non-human animal genomes are female.

In another aspect, provided are methods for making any of the abovenon-human animals. Some such methods comprise: (a) contacting the genomeof a non-human animal pluripotent cell that is not a one-cell stageembryo with: (i) an exogenous repair template comprising an insertnucleic acid flanked by a 5′ homology arm that hybridizes to a 5′ targetsequence at the Slc30a8 locus and a 3′ homology arm that hybridizes to a3′ target sequence at the Slc30a8 locus, wherein the insert nucleic acidcomprises the mutation; and (ii) a Cas9 protein; and (iii) a guide RNAthat hybridizes to a guide RNA recognition sequence within the Slc30a8locus, wherein the Slc30a8 gene is modified to comprise the mutation;and (b) introducing the modified non-human animal pluripotent cell intoa host embryo; and (c) implanting the host embryo into a surrogatemother to produce a genetically modified F0 generation non-human animalin which the Slc30a8 gene is modified to comprise the mutation, whereinthe mutation results in the F0 generation non-human animal having anenhanced capacity for insulin secretion relative to a non-human animalwithout the mutation when fed the high-fat diet. Some such methodscomprise: (a) contacting the genome of a non-human animal pluripotentcell that is not a one-cell stage embryo with: (i) an exogenous repairtemplate comprising an insert nucleic acid flanked by a 5′ homology armthat hybridizes to a 5′ target sequence at the Slc30a8 locus and a 3′homology arm that hybridizes to a 3′ target sequence at the Slc30a8locus, wherein the insert nucleic acid comprises the mutation; and (ii)a Cas9 protein; and (iii) a guide RNA that hybridizes to a guide RNArecognition sequence within the Slc30a8 locus, wherein the Slc30a8 geneis modified to comprise the mutation; and (b) introducing the modifiednon-human animal pluripotent cell into a host embryo; and (c) gestatingthe host embryo in a surrogate mother to produce a genetically modifiedF0 generation non-human animal in which the Slc30a8 gene is modified tocomprise the mutation, wherein the mutation results in the F0 generationnon-human animal having an enhanced capacity for insulin secretionrelative to a non-human animal without the mutation when fed thehigh-fat diet. Optionally, the pluripotent cell is an embryonic stemcell. Optionally, the exogenous repair template is a large targetingvector that is at least 10 kb in length, or wherein the exogenous repairtemplate is a large targeting vector in which the sum total of the 5′homology arm and the 3′ homology arm is at least 10 kb in length.

Some such methods comprise: (a) contacting the genome of a non-humananimal one-cell stage embryo with: (i) an exogenous repair templatecomprising an insert nucleic acid flanked by a 5′ homology arm thathybridizes to a 5′ target sequence at the Slc30a8 locus and a 3′homology arm that hybridizes to a 3′ target sequence at the Slc30a8locus, wherein the insert nucleic acid comprises the mutation; (ii) aCas9 protein; and (iii) a guide RNA that that hybridizes to a guide RNArecognition sequence within the Slc30a8 locus, wherein the Slc30a8 geneis modified to comprise the mutation; and (b) implanting the modifiednon-human animal one-cell stage embryo into a surrogate mother toproduce a genetically modified F0 generation non-human animal in whichthe Slc30a8 gene is modified to comprise the mutation, wherein themutation results in the F0 generation non-human animal having anenhanced capacity for insulin secretion relative to a non-human animalwithout the mutation when fed the high-fat diet. Some such methodscomprise: (a) contacting the genome of a non-human animal one-cell stageembryo with: (i) an exogenous repair template comprising an insertnucleic acid flanked by a 5′ homology arm that hybridizes to a 5′ targetsequence at the Slc30a8 locus and a 3′ homology arm that hybridizes to a3′ target sequence at the Slc30a8 locus, wherein the insert nucleic acidcomprises the mutation; (ii) a Cas9 protein; and (iii) a guide RNA thatthat hybridizes to a guide RNA recognition sequence within the Slc30a8locus, wherein the Slc30a8 gene is modified to comprise the mutation;and (b) gestating the modified non-human animal one-cell stage embryo ina surrogate mother to produce a genetically modified F0 generationnon-human animal in which the Slc30a8 gene is modified to comprise themutation, wherein the mutation results in the F0 generation non-humananimal having an enhanced capacity for insulin secretion relative to anon-human animal without the mutation when fed the high-fat diet.

In some such methods, step (a) further comprises contacting the genomeof the non-human animal pluripotent cell or the non-human one-cell stageembryo with a second guide RNA that hybridizes to a second guide RNArecognition sequence within the Slc30a8 locus.

In some such methods, the exogenous repair template a single-strandedoligodeoxynucleotide. Optionally, the exogenous repair template isbetween about 80 nucleotides to about 200 nucleotides in length.

In another aspect, provided are methods of screening compounds foractivity for ameliorating or exacerbating type-2-diabetes. Some suchmethods comprise: (a) contacting any of the above subject non-humananimals with the compound; and (b) measuring one or more of thefollowing in the subject non-human animal relative to a controlnon-human animal not contacted with the compound, wherein the controlnon-human animal comprises the same Slc30a8 mutation as the subjectnon-human animal: (1) glucose-induced insulin secretion when fed thehigh-fat diet; (2) pancreatic beta-cell proliferation levels when fedthe high-fat diet; (3) number of pancreatic beta cells when fed thehigh-fat diet; and (4) fed plasma insulin levels after blockade of theinsulin receptor, whereby activity for ameliorating type 2 diabetes isidentified by one or more of the following in the subject non-humananimal compared with the control non-human animal: (1) increasedglucose-induced insulin secretion when fed the high-fat diet; (2)increased pancreatic beta-cell proliferation when fed the high-fat diet;(3) increased number of pancreatic beta cells when fed the high-fatdiet; and (4) increased fed plasma insulin levels after blockade of theinsulin receptor, and whereby activity for exacerbating type 2 diabetesis identified by one or more of the following in the subject non-humananimal compared with the control non-human animal: (1) decreasedglucose-induced insulin secretion when fed the high-fat diet; (2)decreased pancreatic beta-cell proliferation when fed the high-fat diet;(3) decreased number of pancreatic beta cells when fed the high-fatdiet; and (4) decreased fed plasma insulin levels after blockade of theinsulin receptor.

Some such methods comprise: (a) contacting any of the above subjectnon-human animals with the compound; and (b) measuring one or more ofthe following in the subject non-human animal relative to a controlnon-human animal not contacted with the compound, wherein the controlnon-human animal comprises the same Slc30a8 mutation as the subjectnon-human animal: (1) capacity to secrete insulin in response tohyperglycemia; (2) insulin clearance; (3) mitochondrial gene expression;and (4) Hvcn1 expression, whereby activity for ameliorating type 2diabetes is identified by one or more of the following in the subjectnon-human animal compared with the control non-human animal: (1)increased capacity to secrete insulin in response to hyperglycemia; (2)increased insulin clearance; (3) decreased mitochondrial geneexpression; and (4) increased Hvcn1 expression, and whereby activity forexacerbating type 2 diabetes is identified by one or more of thefollowing in the subject non-human animal compared with the controlnon-human animal: (1) decreased capacity to secrete insulin in responseto hyperglycemia; (2) decreased insulin clearance; (3) increasedmitochondrial gene expression; and (4) decreased Hvcn1 expression.

In another aspect, provided are non-human animal cells whose genomecomprises an endogenous Slc30a8 locus comprising a mutated Slc30a8 gene,wherein the mutated Slc30a8 gene encodes a truncated SLC30A8 protein,and wherein a non-human animal comprising the mutated Slc30a8 gene hasan enhanced capacity for insulin secretion relative to a non-humananimal without the mutation.

In another aspect, provided are non-human animal genomes comprising anendogenous Slc30a8 locus comprising a mutated Slc30a8 gene, wherein themutated Slc30a8 gene encodes a truncated SLC30A8 protein, and wherein anon-human animal comprising the mutated Slc30a8 gene has an enhancedcapacity for insulin secretion relative to a non-human animal withoutthe mutation.

In another aspect, provided are targeting vectors for generating amutated Slc30a8 gene at an endogenous Slc30a8 locus in a non-humananimal, wherein the targeting vector comprises a 5′ homology armtargeting a 5′ target sequence at the endogenous Slc30a8 locus and a 3′homology arm targeting a 3′ target sequence at the endogenous Slc30a8locus, wherein the targeting vector comprises a mutation in the Slc30a8gene, wherein the mutated Slc30a8 gene encodes a truncated SLC30A8protein, and wherein a non-human animal comprising the mutated Slc30a8gene has an enhanced capacity for insulin secretion relative to anon-human animal without the mutation.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D show analysis of Slc30a8 RNA and protein in islets from maleR138X mice on chow diet. FIG. 1A shows Slc30a8 RNA in situ hybridizationof pancreatic islets isolated from wild type (WT), homozygous knockout(KO), and homozygous R138X mice. KO islets were used as negativecontrol. Red: glucagon RNA, green: insulin RNA, white: Slc30a8 RNA. FIG.1B shows quantification of islet Slc30a8 RNA levels using TAQMAN®analysis. FIG. 1C shows Western blot of islets isolated from chow-fedWT, KO and R138X mice. KO islets were used as negative control. Arrow:SLC30A8 protein; * denotes unspecific bands. FIG. 1D shows dithizonestaining of pancreatic islets isolated from WT, KO, and R138X mice.

FIGS. 2A-2P show the metabolic phenotype of male homozygous R138X miceon chow diet. FIGS. 2A-2D and 2L show body weight (FIG. 2L), bloodglucose (FIG. 2A), plasma insulin (FIG. 2B), proinsulin (FIG. 2C), andC-peptide (FIG. 2D) levels in fed and fasted WT and R138X mice. FIGS.2E-2F show proinsulin/insulin ratio (FIG. 2E) and C-peptide/insulinratio (FIG. 2F) in fed and fasted WT and R138X mice. FIG. 2G shows oralglucose tolerance test in WT and R138X mice. Data are displayed as bloodglucose over time. FIG. 2H shows plasma insulin levels in WT or R138Xmice after an overnight fast (time 0) and at the indicated times afteran intraperitoneal injection of glucose. Data are displayed as bloodglucose levels over time. FIG. 2I shows histology for insulin inpancreas isolated from WT and R138X mice. FIG. 2J shows quantificationof pancreatic insulin staining. FIG. 2K shows quantification of isletnumber. Values represent the means±SEM (n=6-20 mice per genotype). FIG.2M shows results of an insulin tolerance test in 4 hr fasted WT andR138X mice. Data are displayed as blood glucose levels over time(n=6/genotype). FIG. 2N shows further quantification of pancreaticinsulin staining (islet mass; n=6/genotype). Values represent themeans±SEM. FIGS. 2O-2P show proinsulin/C-peptide ratio (FIG. 2O) andinsulin/C-peptide ratio (FIG. 2P) in fed and fasted WT and R138X mice ona chow diet. N=18-20 mice per genotype.

FIGS. 3A-3M show the metabolic phenotype of male homozygous R138X miceon HFD diet. FIGS. 3A-3F show blood glucose (FIG. 3A), plasma insulin(FIG. 3B), proinsulin (FIG. 3C), and C-peptide (FIG. 3D) levels in fedand fasted WT and R138X mice. FIGS. 3E-3F show proinsulin/insulin ratio(FIG. 3E) and C-peptide/insulin ratio (FIG. 3F) in fed and fasted WT andR138X mice. FIG. 3G shows oral glucose tolerance test in WT and R138Xmice. Data are displayed as blood glucose over time. Area under thecurve is shown on the right. FIG. 3H shows blood insulin levels in WT orR138X mice after an overnight fast (time 0) and at the indicated timesafter an intraperitoneal injection of glucose. Data are displayed asblood glucose levels over time. FIG. 3I shows histology for insulin inpancreas isolated from WT and R138X mice. FIG. 3J shows quantificationof pancreatic insulin staining. FIG. 3K shows quantification of isletnumber. FIG. 3L shows immunohistochemistry for Ki67 (green) and insulin(red). FIG. 3M shows quantification of Ki67 and insulin double-positivecells. Values represent the means±SEM (n=4-17 mice per genotype).

FIGS. 4A-4K show metabolic phenotype of male homozygous Slc30a8 KO miceon HFD diet. FIGS. 4A-4D show blood glucose (FIG. 4A), plasma insulin(FIG. 4B), proinsulin (FIG. 4C), and C-peptide (FIG. 4D) levels in fedand fasted WT and Slc30a8 KO mice. FIGS. 4E-4F show proinsulin/insulinratio (FIG. 4E) and C-peptide/insulin ratio (FIG. 4F) in fed and fastedWT and Slc30a8 KO mice. FIG. 4G shows oral glucose tolerance test in WTand Slc30a8 KO mice. Data are displayed as blood glucose levels overtime. FIG. 4H shows blood insulin levels in WT or Slc30a8 KO mice afteran overnight fast (time 0) and at the indicated times after anintraperitoneal injection of glucose. Data are displayed as glucoselevels over time. FIG. 4I shows histology for insulin in pancreasisolated from WT and Slc30a8 KO mice. FIG. 4J shows quantification ofpancreatic insulin staining. FIG. 4K shows quantification of isletnumber. Values represent the means±SEM (n=6-17 mice per genotype).

FIG. 5 shows in vitro expression and stabilization of human R138Xprotein by proteasomal inhibition. Western blot analysis of HEK293lysates is shown after overexpression of either myc-tagged SLC30A8 WT orR138X protein alone or together. Cells were treated for 6 hr withindicated compounds and probed with the respective antibodies.

FIGS. 6A-6E show glucagon histology in chow and HFD conditions isunchanged in male homozygous R138X mice. FIG. 6A shows histology forglucagon in pancreas isolated from chow-fed WT and R138X mice. FIGS. 6Band 6E show quantification of pancreatic glucagon staining shown in FIG.6A. Values represent the means±SEM (n=6 per genotype). FIG. 6C showshistology for glucagon in pancreas isolated from HFD-fed WT and R138Xmice. FIG. 6D shows quantification of pancreatic glucagon staining shownin FIG. 6C (n=4-17 mice per genotype).

FIGS. 7A-7K show metabolic phenotype of male homozygous Slc30a8 KO miceon chow diet. FIGS. 7A-7D show blood glucose (FIG. 7A), plasma insulin(FIG. 7B), proinsulin (FIG. 7C), and C-peptide (FIG. 7D) levels in fedand fasted WT and Slc30a8 KO mice. FIGS. 7E-7F show proinsulin/insulinratio (FIG. 7E) and C-peptide/insulin ratio (FIG. 7F) in fed and fastedWT and Slc30a8 KO mice. FIG. 7G shows oral glucose tolerance test in WTand Slc30a8 KO mice. Data are displayed as blood glucose levels overtime. FIG. 7H shows plasma insulin levels in WT or Slc30a8 KO mice afteran overnight fast (time 0) and at the indicated times after anintraperitoneal injection of glucose. Data are displayed as bloodglucose levels over time. FIG. 7I shows histology for insulin inpancreas isolated from WT and Slc30a8 KO mice. FIG. 7J showsquantification of pancreatic insulin staining. FIG. 7K showsquantification of islet number. Values represent the means±SEM (n=6-10mice per genotype).

FIGS. 8A-8K show metabolic phenotype of female homozygous R138X mice onHFD. FIGS. 8A-8D show blood glucose (FIG. 8A), plasma insulin (FIG. 8B),proinsulin (FIG. 8C), and C-peptide (FIG. 8D) levels in fed and fastedWT and R138X mice. FIGS. 8E-8F show proinsulin/insulin ratio (FIG. 8E)and C-peptide/insulin ratio (FIG. 8F) in fed and fasted WT and R138Xmice. FIG. 8G shows oral glucose tolerance test in WT and R138X mice.Data are displayed as glucose levels over time. FIG. 8H shows plasmainsulin levels in WT or R138X mice after an overnight fast (time 0) andat the indicated times after an intraperitoneal injection of glucose.Data are displayed as glucose levels over time. FIG. 8I shows histologyfor insulin in pancreas isolated from WT and R138X mice. FIG. 8J showsquantification of pancreatic insulin staining. FIG. 8K showsquantification of islet number. Values represent the means±SEM (n=6-9mice per genotype).

FIGS. 9A-9M show heterozygous R138X (HET) mice on HFD have higherglucose-stimulated insulin levels. FIG. 9A shows Western blot of isletsisolated from chow-fed WT, heterozygous R138X mice. Arrow: SLC30A8protein; * denotes unspecific bands. FIG. 9B shows dithizone staining ofpancreatic islets isolated from WT and heterozygous R138X mice. FIGS.9C-9F show blood glucose (FIG. 9C), plasma insulin (FIG. 9D), proinsulin(FIG. 9E), and C-peptide (FIG. 9F) levels in fed and fasted WT and R138XHET mice. FIGS. 9G-9H show proinsulin/insulin ratio (FIG. 9G) andC-peptide/insulin ratio (FIG. 9H) in fed and fasted WT and HET mice.FIG. 9I shows oral glucose tolerance test in WT and R138X HET mice. Dataare displayed as blood glucose levels over time. FIG. 9J shows plasmainsulin levels in WT or R138X HET mice after an overnight fast (time 0)and at the indicated times after an intraperitoneal injection ofglucose. Data are displayed as glucose levels over time. FIG. 9K showshistology for insulin in pancreas isolated from WT and R138X HET mice.FIG. 9L shows quantification of pancreatic insulin staining. FIG. 9Mshows quantification of islet number. Values represent the means±SEM(n=6-9 mice per genotype).

FIGS. 10A-10C show schematics for targeting the mouse Slc30a8 locus togenerate R138X mice. FIG. 10A shows the wild type mouse locus andindicates the location of the pArg137* C>T point mutation and the 29 bpdeletion that are generated through targeting. An allele-specificscreening assay (8084 AS) and a TAQMAN® downstream screening assay (8084TD) are shown. FIG. 10B shows a schematic of the targeted locus. FIG.10C shows a schematic of the targeted locus after self-excising of theself-deleting drug selection cassette.

FIG. 11 shows a protein alignment of the wild type mouse SLC30A8 protein(SEQ ID NO: 12), the expected truncated SLC30A8 Arg137* protein in R138Xmice (SEQ ID NO: 13), and the wild type human SLC30A8 protein (SEQ IDNO: 14).

FIG. 12 shows fed plasma glucose levels and fed plasma insulin levels inWT (closed symbols) and homozygous R138X mice (open symbols) treatedwith either S961 (dashed lines) or PBS (solid lines) using a constantminipump infusion. Fed plasma glucose levels and insulin levels weremeasured at the indicated times throughout the experiment (21 days).

FIGS. 13A-13I show that R138X mice secrete more insulin under chronichyperglycemia caused by the insulin receptor antagonist S961. FIG. 13Ashows nonfasted plasma glucose levels in R138X and WT mice continuouslytreated with the insulin receptor antagonist S961 (20 nMol/week) or PBSfor 22 days. FIG. 13B shows nonfasted plasma insulin on days 0, 4, 19and 22 R138X and WT mice treated with S961 (20 nMol/week) or PBS. FIG.13C shows plasma active GLP-1 levels in WT and R138X mice after 22 daysof treatment. FIGS. 13D and 13E show fed and fasted glucose (FIG. 13D)and insulin (FIG. 13E) levels measured day 19 and 20 after initiation oftreatment. FIG. 13F shows immunohistochemistry for KI-67 (white),insulin (green), and glucagon (red). FIG. 13G shows quantification ofKI-67 and insulin double-positive cells. FIG. 13H shows quantificationof pancreatic insulin staining shown in (FIG. 13I). FIG. 13I showshistology for insulin in pancreas isolated from WT and R138X mice after22 days of treatment. Values represent the means±SEM (n=5-7 mice pertreatment and genotype).

FIGS. 14A-14G show hormone levels and glucagon histology after 22 daysof insulin receptor antagonist S961 (20 nMol/week) or PBS S961treatment. Insulin (FIG. 14A), Proinsulin (FIG. 14B), C-peptide (FIG.14C), Proinsulin/C-peptide ratio (FIG. 14D), Insulin/C-peptide ratio(FIG. 14E) in fed and fasted WT and R138X mice after 22 days oftreatment. FIG. 14F shows quantification of pancreatic glucagon stainingshown in FIG. 14G. FIG. 14G shows histology for glucagon in pancreasisolated from WT and R138X mice after 22 days of treatment. Valuesrepresent the means±SEM (n=5-7 mice per genotype).

FIG. 15 shows gene expression of Slc30a8, expressed in RPKM (reads perkilobase million).

FIGS. 16A-16C show gene expression changes in islets from WT versusR138X mice. FIG. 16A shows RPKM values for insulin 2 (Ins2) and insulin1 (Ins1). FIG. 16B shows RPKM values for beta-cell regulators. FIG. 16Cshows RPKM values for Hvcn1.

FIG. 17 shows circulatory zinc levels in fed and fasted male WT andhomozygous R138X mice after 20 weeks on high-fat diet (HFD). The micewere approximately 29 weeks of age. 12 WT mice were assessed, and 13R138X mice were assessed.

DEFINITIONS

The terms “protein,” “polypeptide,” and “peptide,” used interchangeablyherein, include polymeric forms of amino acids of any length, includingcoded and non-coded amino acids and chemically or biochemically modifiedor derivatized amino acids. The terms also include polymers that havebeen modified, such as polypeptides having modified peptide backbones.The term “domain” refers to any part of a protein or polypeptide havinga particular function or structure.

Proteins are said to have an “N-terminus” and a “C-terminus.” The term“N-terminus” relates to the start of a protein or polypeptide,terminated by an amino acid with a free amine group (—NH2). The term“C-terminus” relates to the end of an amino acid chain (protein orpolypeptide), terminated by a free carboxyl group (—COOH).

The terms “nucleic acid” and “polynucleotide,” used interchangeablyherein, include polymeric forms of nucleotides of any length, includingribonucleotides, deoxyribonucleotides, or analogs or modified versionsthereof. They include single-, double-, and multi-stranded DNA or RNA,genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purinebases, pyrimidine bases, or other natural, chemically modified,biochemically modified, non-natural, or derivatized nucleotide bases.

Nucleic acids are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. An end of an oligonucleotide is referred to as the “5′ end” ifits 5′ phosphate is not linked to the 3′ oxygen of a mononucleotidepentose ring. An end of an oligonucleotide is referred to as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of anothermononucleotide pentose ring. A nucleic acid sequence, even if internalto a larger oligonucleotide, also may be said to have 5′ and 3′ ends. Ineither a linear or circular DNA molecule, discrete elements are referredto as being “upstream” or 5′ of the “downstream” or 3′ elements.

The term “genomically integrated” refers to a nucleic acid that has beenintroduced into a cell such that the nucleotide sequence integrates intothe genome of the cell. Any protocol may be used for the stableincorporation of a nucleic acid into the genome of a cell.

The term “targeting vector” refers to a recombinant nucleic acid thatcan be introduced by homologous recombination,non-homologous-end-joining-mediated ligation, or any other means ofrecombination to a target position in the genome of a cell.

The term “wild type” includes entities having a structure and/oractivity as found in a normal (as contrasted with mutant, diseased,altered, or so forth) state or context. Wild type genes and polypeptidesoften exist in multiple different forms (e.g., alleles).

The term “endogenous sequence” refers to a nucleic acid sequence thatoccurs naturally within a cell or non-human animal. For example, anendogenous Slc30a8 sequence of a non-human animal refers to a nativeSlc30a8 sequence that naturally occurs at the Slc30a8 locus in thenon-human animal. The term “endogenous locus” refers to a location on achromosome at which a particular genetic element is naturally found. Forexample, an endogenous Slc30a8 locus of a non-human animal refers to thenaturally occurring Slc30a8 locus in the non-human animal (i.e., theregion of the non-human animal genome at which the Slc30a8 gene isnaturally found). As an example, the endogenous mouse Slc30a8 locus mapsto chromosome 15 (NCBI RefSeq GeneID 239436; Assembly GRCm38.p4;location NC_000081.6 (52295553-52335733)). The term “endogenous Slc30a8locus” of a non-human animal does not refer to a Slc30a8 gene randomlyinserted into the genome of the non-human animal or inserted at a regionother than the region of the non-human animal genome at which theSlc30a8 gene is naturally found. Likewise, the term “endogenous Slc30a8locus” of a non-human animal does not refer to a Slc30a8 gene located,for example, in cells (e.g., human beta cells) grafted into thenon-human animal.

“Exogenous” molecules or sequences include molecules or sequences thatare not normally present in a cell in that form. Normal presenceincludes presence with respect to the particular developmental stage andenvironmental conditions of the cell. An exogenous molecule or sequence,for example, can include a mutated version of a corresponding endogenoussequence within the cell or can include a sequence corresponding to anendogenous sequence within the cell but in a different form (i.e., notwithin a chromosome). In contrast, endogenous molecules or sequencesinclude molecules or sequences that are normally present in that form ina particular cell at a particular developmental stage under particularenvironmental conditions.

The term “heterologous” when used in the context of a nucleic acid or aprotein indicates that the nucleic acid or protein comprises at leasttwo segments that do not naturally occur together in the same molecule.For example, the term “heterologous,” when used with reference tosegments of a nucleic acid or segments of a protein, indicates that thenucleic acid or protein comprises two or more sub-sequences that are notfound in the same relationship to each other (e.g., joined together) innature. As one example, a “heterologous” region of a nucleic acid vectoris a segment of nucleic acid within or attached to another nucleic acidmolecule that is not found in association with the other molecule innature. For example, a heterologous region of a nucleic acid vectorcould include a coding sequence flanked by sequences not found inassociation with the coding sequence in nature. Likewise, a“heterologous” region of a protein is a segment of amino acids within orattached to another peptide molecule that is not found in associationwith the other peptide molecule in nature (e.g., a fusion protein, or aprotein with a tag). Similarly, a nucleic acid or protein can comprise aheterologous label or a heterologous secretion or localization sequence.

“Codon optimization” takes advantage of the degeneracy of codons, asexhibited by the multiplicity of three-base pair codon combinations thatspecify an amino acid, and generally includes a process of modifying anucleic acid sequence for enhanced expression in particular host cellsby replacing at least one codon of the native sequence with a codon thatis more frequently or most frequently used in the genes of the host cellwhile maintaining the native amino acid sequence. For example, a nucleicacid encoding a Cas9 protein can be modified to substitute codons havinga higher frequency of usage in a given prokaryotic or eukaryotic cell,including a bacterial cell, a yeast cell, a human cell, a non-humancell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, ahamster cell, or any other host cell, as compared to the naturallyoccurring nucleic acid sequence. Codon usage tables are readilyavailable, for example, at the “Codon Usage Database.” These tables canbe adapted in a number of ways. See Nakamura et al. (2000) Nucleic AcidsResearch 28:292, herein incorporated by reference in its entirety forall purposes. Computer algorithms for codon optimization of a particularsequence for expression in a particular host are also available (see,e.g., Gene Forge).

The term “locus” refers to a specific location of a gene (or significantsequence), DNA sequence, polypeptide-encoding sequence, or position on achromosome of the genome of an organism. For example, a “Slc30a8 locus”may refer to the specific location of a Slc30a8 gene, Slc30a8 DNAsequence, SLC30a8-encoding sequence, or Slc30a8 position on a chromosomeof the genome of an organism that has been identified as to where such asequence resides. A “Slc30a8 locus” may comprise a regulatory element ofa Slc30a8 gene, including, for example, an enhancer, a promoter, 5′and/or 3′ untranslated region (UTR), or a combination thereof.

The term “gene” refers to a DNA sequence in a chromosome that codes fora product (e.g., an RNA product and/or a polypeptide product) andincludes the coding region interrupted with non-coding introns andsequence located adjacent to the coding region on both the 5′ and 3′ends such that the gene corresponds to the full-length mRNA (includingthe 5′ and 3′ untranslated sequences). The term “gene” also includesother non-coding sequences including regulatory sequences (e.g.,promoters, enhancers, and transcription factor binding sites),polyadenylation signals, internal ribosome entry sites, silencers,insulating sequence, and matrix attachment regions. These sequences maybe close to the coding region of the gene (e.g., within 10 kb) or atdistant sites, and they influence the level or rate of transcription andtranslation of the gene.

The term “allele” refers to a variant form of a gene. Some genes have avariety of different forms, which are located at the same position, orgenetic locus, on a chromosome. A diploid organism has two alleles ateach genetic locus. Each pair of alleles represents the genotype of aspecific genetic locus. Genotypes are described as homozygous if thereare two identical alleles at a particular locus and as heterozygous ifthe two alleles differ.

The “coding region” or “coding sequence” of a gene consists of theportion of a gene's DNA or RNA, composed of exons, that codes for aprotein. The region begins at the start codon on the 5′ end and ends atthe stop codon on the 3′ end.

A “promoter” is a regulatory region of DNA usually comprising a TATA boxcapable of directing RNA polymerase II to initiate RNA synthesis at theappropriate transcription initiation site for a particularpolynucleotide sequence. A promoter may additionally comprise otherregions which influence the transcription initiation rate. The promotersequences disclosed herein modulate transcription of an operably linkedpolynucleotide. A promoter can be active in one or more of the celltypes disclosed herein (e.g., a eukaryotic cell, a non-human mammaliancell, a human cell, a rodent cell, a pluripotent cell, a one-cell stageembryo, a differentiated cell, or a combination thereof). A promoter canbe, for example, a constitutively active promoter, a conditionalpromoter, an inducible promoter, a temporally restricted promoter (e.g.,a developmentally regulated promoter), or a spatially restrictedpromoter (e.g., a cell-specific or tissue-specific promoter). Examplesof promoters can be found, for example, in WO 2013/176772, hereinincorporated by reference in its entirety for all purposes.

“Operable linkage” or being “operably linked” includes juxtaposition oftwo or more components (e.g., a promoter and another sequence element)such that both components function normally and allow the possibilitythat at least one of the components can mediate a function that isexerted upon at least one of the other components. For example, apromoter can be operably linked to a coding sequence if the promotercontrols the level of transcription of the coding sequence in responseto the presence or absence of one or more transcriptional regulatoryfactors. Operable linkage can include such sequences being contiguouswith each other or acting in trans (e.g., a regulatory sequence can actat a distance to control transcription of the coding sequence).

The term “variant” refers to a nucleotide sequence differing from thesequence most prevalent in a population (e.g., by one nucleotide) or aprotein sequence different from the sequence most prevalent in apopulation (e.g., by one amino acid).

“Sequence identity” or “identity” in the context of two polynucleotidesor polypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins, residue positions which are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known. Typically, this involves scoring aconservative substitution as a partial rather than a full mismatch,thereby increasing the percentage sequence identity. Thus, for example,where an identical amino acid is given a score of 1 and anon-conservative substitution is given a score of zero, a conservativesubstitution is given a score between zero and 1. The scoring ofconservative substitutions is calculated, e.g., as implemented in theprogram PC/GENE (Intelligenetics, Mountain View, Calif.).

“Percentage of sequence identity” includes the value determined bycomparing two optimally aligned sequences (greatest number of perfectlymatched residues) over a comparison window, wherein the portion of thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) as compared to the reference sequence (whichdoes not comprise additions or deletions) for optimal alignment of thetwo sequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid base or amino acid residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison, and multiplying the result by 100to yield the percentage of sequence identity. Unless otherwise specified(e.g., the shorter sequence includes a linked heterologous sequence),the comparison window is the full length of the shorter of the twosequences being compared.

Unless otherwise stated, sequence identity/similarity values include thevalue obtained using GAP Version 10 using the following parameters: %identity and % similarity for a nucleotide sequence using GAP Weight of50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; %identity and % similarity for an amino acid sequence using GAP Weight of8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or anyequivalent program thereof. “Equivalent program” includes any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The term “conservative amino acid substitution” refers to thesubstitution of an amino acid that is normally present in the sequencewith a different amino acid of similar size, charge, or polarity.Examples of conservative substitutions include the substitution of anon-polar (hydrophobic) residue such as isoleucine, valine, or leucinefor another non-polar residue. Likewise, examples of conservativesubstitutions include the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, or between glycine and serine. Additionally,the substitution of a basic residue such as lysine, arginine, orhistidine for another, or the substitution of one acidic residue such asaspartic acid or glutamic acid for another acidic residue are additionalexamples of conservative substitutions. Examples of non-conservativesubstitutions include the substitution of a non-polar (hydrophobic)amino acid residue such as isoleucine, valine, leucine, alanine, ormethionine for a polar (hydrophilic) residue such as cysteine,glutamine, glutamic acid or lysine and/or a polar residue for anon-polar residue. Typical amino acid categorizations are summarizedbelow.

TABLE 1 Amino Acid Categorizations. Alanine Ala A Nonpolar Neutral 1.8Arginine Arg R Polar Positive −4.5 Asparagine Asn N Polar Neutral −3.5Aspartic acid Asp D Polar Negative −3.5 Cysteine Cys C Nonpolar Neutral2.5 Glutamic acid Glu E Polar Negative −3.5 Glutamine Gln Q PolarNeutral −3.5 Glycine Gly G Nonpolar Neutral −0.4 Histidine His H PolarPositive −3.2 Isoleucine Ile I Nonpolar Neutral 4.5 Leucine Leu LNonpolar Neutral 3.8 Lysine Lys K Polar Positive −3.9 Methionine Met MNonpolar Neutral 1.9 Phenylalanine Phe F Nonpolar Neutral 2.8 ProlinePro P Nonpolar Neutral −1.6 Serine Ser S Polar Neutral −0.8 ThreonineThr T Polar Neutral −0.7 Tryptophan Trp W Nonpolar Neutral −0.9 TyrosineTyr Y Polar Neutral −1.3 Valine Val V Nonpolar Neutral 4.2

A “homologous” sequence (e.g., nucleic acid sequence) includes asequence that is either identical or substantially similar to a knownreference sequence, such that it is, for example, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the knownreference sequence. Homologous sequences can include, for example,orthologous sequence and paralogous sequences. Homologous genes, forexample, typically descend from a common ancestral DNA sequence, eitherthrough a speciation event (orthologous genes) or a genetic duplicationevent (paralogous genes). “Orthologous” genes include genes in differentspecies that evolved from a common ancestral gene by speciation.Orthologs typically retain the same function in the course of evolution.“Paralogous” genes include genes related by duplication within a genome.Paralogs can evolve new functions in the course of evolution.

The term “in vitro” includes artificial environments and to processes orreactions that occur within an artificial environment (e.g., a testtube). The term “in vivo” includes natural environments (e.g., a cell ororganism or body) and to processes or reactions that occur within anatural environment. The term “ex vivo” includes cells that have beenremoved from the body of an individual and to processes or reactionsthat occur within such cells.

The term “reporter gene” refers to a nucleic acid having a sequenceencoding a gene product (typically an enzyme) that is easily andquantifiably assayed when a construct comprising the reporter genesequence operably linked to an endogenous or heterologous promoterand/or enhancer element is introduced into cells containing (or whichcan be made to contain) the factors necessary for the activation of thepromoter and/or enhancer elements. Examples of reporter genes include,but are not limited, to genes encoding beta-galactosidase (lacZ), thebacterial chloramphenicol acetyltransferase (cat) genes, fireflyluciferase genes, genes encoding beta-glucuronidase (GUS), and genesencoding fluorescent proteins. A “reporter protein” refers to a proteinencoded by a reporter gene.

The term “fluorescent reporter protein” as used herein means a reporterprotein that is detectable based on fluorescence wherein thefluorescence may be either from the reporter protein directly, activityof the reporter protein on a fluorogenic substrate, or a protein withaffinity for binding to a fluorescent tagged compound. Examples offluorescent proteins include green fluorescent proteins (e.g., GFP,GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, Monomeric AzamiGreen, CopGFP, AceGFP, and ZsGreenl), yellow fluorescent proteins (e.g.,YFP, eYFP, Citrine, Venus, YPet, PhiYFP, and ZsYellowl), bluefluorescent proteins (e.g., BFP, eBFP, eBFP2, Azurite, mKalamal, GFPuv,Sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP,Cerulean, CyPet, AmCyanl, and Midoriishi-Cyan), red fluorescent proteins(e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1,DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2,eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins(e.g., mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange,mTangerine, and tdTomato), and any other suitable fluorescent proteinwhose presence in cells can be detected by flow cytometry methods.

Repair in response to double-strand breaks (DSBs) occurs principallythrough two conserved DNA repair pathways: homologous recombination (HR)and non-homologous end joining (NHEJ). See Kasparek & Humphrey (2011)Seminars in Cell & Dev. Biol. 22:886-897, herein incorporated byreference in its entirety for all purposes. Likewise, repair of a targetnucleic acid mediated by an exogenous donor nucleic acid can include anyprocess of exchange of genetic information between the twopolynucleotides.

The term “recombination” includes any process of exchange of geneticinformation between two polynucleotides and can occur by any mechanism.Recombination can occur via homology directed repair (HDR) or homologousrecombination (HR). HDR or HR includes a form of nucleic acid repairthat can require nucleotide sequence homology, uses a “donor” moleculeas a template for repair of a “target” molecule (i.e., the one thatexperienced the double-strand break), and leads to transfer of geneticinformation from the donor to target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or synthesis-dependent strand annealing, in which the donor is usedto resynthesize genetic information that will become part of the target,and/or related processes. In some cases, the donor polynucleotide, aportion of the donor polynucleotide, a copy of the donor polynucleotide,or a portion of a copy of the donor polynucleotide integrates into thetarget DNA. See Wang et al. (2013) Cell 153:910-918; Mandalos et al.(2012) PLOS ONE 7:e45768:1-9; and Wang et al. (2013) Nat Biotechnol.31:530-532, each of which is herein incorporated by reference in itsentirety for all purposes.

NHEJ includes the repair of double-strand breaks in a nucleic acid bydirect ligation of the break ends to one another or to an exogenoussequence without the need for a homologous template. Ligation ofnon-contiguous sequences by NHEJ can often result in deletions,insertions, or translocations near the site of the double-strand break.For example, NHEJ can also result in the targeted integration of anexogenous donor nucleic acid through direct ligation of the break endswith the ends of the exogenous donor nucleic acid (i.e., NHEJ-basedcapture). Such NHEJ-mediated targeted integration can be preferred forinsertion of an exogenous donor nucleic acid when homology directedrepair (HDR) pathways are not readily usable (e.g., in non-dividingcells, primary cells, and cells which perform homology-based DNA repairpoorly). In addition, in contrast to homology-directed repair, knowledgeconcerning large regions of sequence identity flanking the cleavage siteis not needed, which can be beneficial when attempting targetedinsertion into organisms that have genomes for which there is limitedknowledge of the genomic sequence. The integration can proceed vialigation of blunt ends between the exogenous donor nucleic acid and thecleaved genomic sequence, or via ligation of sticky ends (i.e., having5′ or 3′ overhangs) using an exogenous donor nucleic acid that isflanked by overhangs that are compatible with those generated by anuclease agent in the cleaved genomic sequence. See, e.g., US2011/020722, WO 2014/033644, WO 2014/089290, and Maresca et al. (2013)Genome Res. 23(3):539-546, each of which is herein incorporated byreference in its entirety for all purposes. If blunt ends are ligated,target and/or donor resection may be needed to generation regions ofmicrohomology needed for fragment joining, which may create unwantedalterations in the target sequence.

The term “R138X mice” refers to mice carrying a mutation in the mouseSlc30a8 locus corresponding to the human SLC30A8 putative LOF mutation,R138X. The mutation in the mouse Slc30a8 locus is c.409C>T (transcriptaccession number NM_172816.3), p.Arg137X, meaning a C to T substitutionat nucleotide 409 of the coding sequence resulting in the codon encodingamino acid residue 137 being mutated from a codon encoding an arginineresidue to a stop codon (R137*^(stop)). The corresponding human SLC30A8mutation is c.412 C>T (transcript accession number NM_173851),p.Arg138X, meaning a C to T substitution at nucleotide 412 of the codingsequence resulting in the codon encoding amino acid residue 138 beingmutated from a codon encoding an arginine residue to a stop codon(R138*^(stop)). Amino acid residue 137 of the mouse SLC30A8 proteincorresponds with amino acid residue 138 of the human SLC30A8 proteinwhen the two are optimally aligned. See FIG. 11. Although the mutationin the mouse Slc30a8 gene is in the codon encoding amino acid residue137 of the mouse SLC30A8 protein, these mice are referred to herein as“R138X mice” in reference to the corresponding human SLC30A8 mutation.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” a protein maycontain the protein alone or in combination with other ingredients. Thetransitional phrase “consisting essentially of” means that the scope ofa claim is to be interpreted to encompass the specified elements recitedin the claim and those that do not materially affect the basic and novelcharacteristic(s) of the claimed invention. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising.”

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur and that the description includesinstances in which the event or circumstance occurs and instances inwhich it does not.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

The term “and/or” refers to and encompasses any and all possiblecombinations of one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

The term “or” refers to any one member of a particular list and alsoincludes any combination of members of that list.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a protein” or “at least one protein” can include a pluralityof proteins, including mixtures thereof.

Statistically significant means p≤0.05.

DETAILED DESCRIPTION I. Overview

Disclosed herein are non-human animal genomes, non-human animal cells,and non-human animals comprising in their genome a mutated Slc30a8 locusand methods of using such non-human animal cells and non-human animals.The zinc transporter SLC30A8 is primarily expressed in the islets of theendocrine pancreas. SLC30A8 is only one of three genes to date for whichputative loss-of-function mutations have been shown to protect fromdevelopment of type 2 diabetes. This is an interesting but also puzzlingobservation since mouse models deficient in Slc30a8 have mildly impairedglucose homeostasis. We have generated a mouse model comprising amutated Slc30a8 locus mimicking a protective human SLC30A8 allele. Thismouse model can for the first time explain why humans are protected fromtype 2 diabetes. The mice have increased insulin secretory capacity. Inparticular, the mice have higher glucose-induced insulin secretion,which may be mediated in part by an increased number of pancreatic betacells. These SLC30A8 mutation data support the notion that beta-cellexpansion is a valid therapeutic strategy to manage type 2 diabetes.

The cells and non-human animal models disclosed herein can be used fordrug screening as well as to provide insights into the mechanism of T2D(e.g., the mechanism by which SLC30A8 contributes to insulin processingand diabetes) and potentially new therapeutic and diagnostic targets.

II. Non-Human Animals Comprising a Mutated Slc30a8 Locus

The cells and non-human animals disclosed herein comprise (e.g., intheir genome) a mutation (e.g., a mutation that does not naturally occurin non-human animals) in the Slc30a8 locus. The mutated Slc30a8 locuscan have a premature termination codon and/or can encode a truncatedSLC30A8 protein. The non-human animals comprising a mutated Slc30a8locus have an enhanced capacity for insulin secretion when fed ahigh-fat diet relative to a non-human animal without the mutation whenfed the same diet.

A. Slc30a8

The cells and non-human animals described herein comprise a mutatedSlc30a8 (solute carrier family 30 member 8) locus. Other names forSLC30A8 include zinc transporter 8, ZnT-8, and Znt8. The protein encodedby Slc30a8 is a zinc transporter involved in the accumulation of zinc inintracellular vesicles. Slc30a8 is expressed at a high level in thepancreas, particularly in islets of Langerhans. The encoded proteinco-localizes with insulin in the secretory pathway granules of theinsulin-secreting INS-1 cells.

Human SLC30A8 maps to human 8q24.11 on chromosome 8 (NCBI RefSeq GeneID169026; Assembly GRCh38.p7; location 116950273-117176714). The gene hasbeen reported to have eight exons and seven introns. However, isoformswith more than eight exons (including non-coding exons) are alsopossible. The wild type human SLC30A8 protein has been assigned UniProtaccession number Q8IWU4. Two isoforms are known, Q8IWU4 isoform 1 (SEQID NO: 14) and Q8IWU4 isoform 2. The mRNA encoding isoform 1 is assignedNCBI RefSeq NM_173851.2 (SEQ ID NO: 17). The full-length human proteinhas 369 amino acids including six transmembrane regions, fourintracellular topological domains, and three extracellular topologicaldomains. Delineations between these domains are as designated inUniProt. Reference to human SCL30A8 includes the canonical (wild type)forms as well as all allelic forms and isoforms. Any other forms ofhuman SLC30A8 have amino acids numbered for maximal alignment with thewild type form, aligned amino acids being designated the same number.

Mouse Slc30a8 maps to chromosome 15 (NCBI RefSeq GeneID 239436; AssemblyGRCm38.p4; location NC_000081.6 (52295553-52335733)). The gene has beenreported to have eight exons and seven introns. The wild type mouseSLC30A8 protein has been assigned UniProt accession number Q8BGG0 (SEQID NO: 12). The mRNA encoding mouse SLC30A8 is assigned NCBI RefSeqNM_172816.3 (SEQ ID NO: 16). The full-length mouse protein has 367 aminoacids including six transmembrane regions, four intracellulartopological domains, and three extracellular topological domains.Delineations between these domains are as designated in UniProt.Reference to mouse SCL30A8 includes the canonical (wild type) forms, aswell as all allelic forms and isoforms. Any other forms of mouse SLC30A8have amino acids numbered for maximal alignment with the wild type form,aligned amino acids being designated the same number.

An exemplary rat SLC30A8 protein is designated by UniProt AccessionNumber P0CE46.

Polymorphisms in the human SLC30A8 gene are associated with altered riskof type 2 diabetes (T2D). See, e.g., Sladek et al. (2007) Nature445(7130):881-885 and Davidson et al. (2014) Trends Endocrinol. Metab.25(8):415-424, each of which is herein incorporated by reference in itsentirety for all purposes. Some such polymorphisms or mutations inhumans can reduce risk of T2D or are protective against T2D in humans. Apolymorphism or mutation in the SLC30A8 gene that reduces risk of T2Dincludes a polymorphism or mutation that confers protection against T2D,that confers resistance to T2D, or that is associated with protectionagainst or resistance to T2D. For example, various stop codon mutations,frameshift mutations, splice site mutations, or initiator codonvariations that cause protein truncation in human SLC30A8 are protectiveagainst T2D when heterozygous. One of these is designated c.412 C>T(transcript accession number NM_173851), p.Arg138X meaning a C to Tsubstitution at nucleotide 412 of the coding sequence resulting in thecodon encoding amino acid residue 138 being mutated from a codonencoding an arginine residue to a stop codon (R138*^(stop)). See, e.g.,Flannick et al. (2014) Nat. Genet. 46(4):357-363, herein incorporated byreference in its entirety for all purposes. These phenotypes have notbeen replicated in previous animal models but are replicated in thenon-human animals disclosed herein.

B. Mutated Slc30a8 Loci

The mutated Slc30a8 loci disclosed herein result in non-human animalshaving an enhanced capacity for insulin secretion when fed a high-fatdiet relative to a non-human animal without the mutation when fed thesame diet. The mutated Slc30a8 loci may reduce T2D risk. Non-humananimals comprising a mutated Slc30a8 locus can have one or more or allof the following characteristics relative to the non-human animalwithout the mutation (e.g., a wild type non-human animal): (1) increasedglucose-induced insulin secretion when fed a high-fat diet; (2)increased pancreatic beta-cell proliferation when fed a high-fat diet;(3) increased number of pancreatic beta cells when fed a high-fat diet;and (4) increased fed plasma insulin levels after blockade of theinsulin receptor (e.g., with S961). Additionally or alternatively,non-human animals comprising a mutated Slc30a8 locus can have one ormore or all of the following characteristics relative to the non-humananimal without the mutation (e.g., a wild type non-human animal): (1)increased circulating insulin levels after 20, 25, 30, 35, 40, 45, or 50weeks (e.g., after 20 or 30 weeks) being fed on a high-fat diet; (2)increased number of pancreatic beta cells after 20, 25, 30, 35, 40, 45,or 50 weeks (e.g., after 20 or 30 weeks) being fed on a high-fat diet;(3) decrease in the proinsulin-to-insulin ratio when fed a high-fatdiet; (4) increased fed plasma insulin levels after blockade of theinsulin receptor (e.g., after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days); and (5)pancreatic beta cells with improved insulin processing and better health(e.g., as evidenced by decrease in ratio of proinsulin-to-insulin whenfed a high-fat diet). Additionally or alternatively, non-human animalscomprising a mutated Slc30a8 locus can have significant Slc30a8 mRNAexpression in islets (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% ofwild type levels, such as at least 25% or 30% of wild type levels).

Compared to non-human animals without the mutation (e.g., wild typenon-human animals), non-human animals with a mutated Slc30a8 locus canhave a normal metabolic phenotype when fed a control chow diet. Comparedto non-human animals without the mutation (e.g., wild type non-humananimals), non-human animals with a mutated Slc30a8 locus can have normalglucose homeostasis and/or normal glucose-induced insulin secretion whenfed a control chow diet. Compared to non-human animals without themutation (e.g., wild type non-human animals), non-human animals with amutated Slc30a8 locus can also have one or more or all of the followingcharacteristics when fed a control chow diet: do not differsignificantly in body weight, do not differ significantly in circulatingblood glucose levels, do not differ significantly in circulating insulinlevels, do not differ significantly in circulating proinsulin levels, donot differ significantly in circulating C-peptide levels, do not differsignificantly in the ratio of proinsulin/C-peptide, do not differsignificantly in glucose tolerance, do not differ significantly ininsulin sensitivity, do not differ significantly in glucose-inducedinsulin secretion, do not differ significantly in beta-cell mass, and donot differ significantly in alpha-cell mass. Compared to non-humananimals without the mutation (e.g., wild type non-human animals),non-human animals with a mutated Slc30a8 locus can have a decrease inthe ratio of insulin/C-peptide when fed a control chow diet. This mayindicate higher insulin clearance.

Compared to non-human animals without the mutation (e.g., wild typenon-human animals), non-human animals with a mutated Slc30a8 locus havean increased capacity to secrete insulin. As one example, the non-humananimals can have an enhanced capacity for insulin secretion when fed ahigh-fat diet. Such non-human animals can have increased insulinsecretion relative to the non-human animal without the mutation when feda high-fat diet, wherein the increased insulin secretion is associatedwith increased beta-cell proliferation or beta-cell mass relative to thenon-human animal without the mutation. The increased beta-cellproliferation can be insulin-receptor-dependent (e.g., dependent on aninsulin receptor in the beta cells). As another example, the non-humananimals can have an enhanced capacity for insulin secretion in responseto hyperglycemia, such as severe hyperglycemia. Such non-human animalscan have increased insulin secretion in response to hyperglycemiainduced by insulin receptor inhibition relative to the non-human animalwithout the mutation. For example, such non-human animals can haveincreased insulin secretion relative to the non-human animal without themutation in response to hyperglycemia induced by insulin receptorinhibition, wherein the increased insulin secretion is not associatedwith increased beta-cell proliferation or beta-cell mass relative to thenon-human animal without the mutation. The increased insulin secretioncompared to non-human animals without the mutation can be at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. For example, thenon-human animals can have increased insulin secretion in response tohyperglycemia induced by insulin receptor inhibition, such ashyperglycemia caused by the insulin receptor antagonist S961. In somenon-human animals, this effect is not associated with enhanced beta-cellproliferation or increased beta-cell mass. In some non-human animals,the increase in plasma insulin is not a result of one or more or all ofthe following: improved proinsulin processing, decreased insulinclearance, increased beta-cell proliferation, increased islet mass,increased beta-cell mass, increased alpha-cell mass, or insulindepletion in beta cells.

Compared to non-human animals without the mutation (e.g., wild typenon-human animals), non-human animals with a mutated Slc30a8 locus canhave decreased mitochondrial gene expression (e.g., decrease inexpression of genes in the mitochondrial oxidative phosphorylation(OXPHOS) system). Examples of such genes are described in more detail inExample 2 and in Tables 7-9. Compared to non-human animals without themutation (e.g., wild type non-human animals), non-human animals with amutated Slc30a8 locus can have increased Hvcn1 (hydrogen voltage-gatedchannel 1, or voltage-gated hydrogen channel 1) expression.

Compared to non-human animals without the mutation (e.g., wild typenon-human animals), non-human animals with a mutated Slc30a8 locus canhave loss of islet zinc accumulation.

Compared to non-human animals without the mutation (e.g., wild typenon-human animals), non-human animals with a mutated Slc30a8 locus canalso have one or more or all of the following characteristics when fed acontrol chow diet: do not differ in body weight, blood glucose, insulin,or C-peptide levels (fed or fasted); have no change in fed proinsulinlevels; might have decreased fasted proinsulin levels; have no change inthe ratio of proinsulin to insulin (fed or fasted); have an increasedfed ratio of C-peptide to insulin; have no change in the fasted ratio ofC-peptide to insulin; and have no change in glucose tolerance orglucose-induced insulin secretion.

Compared to non-human animals without the mutation (e.g., wild typenon-human animals), non-human animals with a mutated Slc30a8 locus canalso have one or more or all of the following characteristics when fed ahigh-fat diet: have no change in fed or fasted blood glucose; haveincreased fed circulating insulin levels; have no change in fastedcirculating insulin levels; have no change in fed proinsulin levels;have decreased fasted proinsulin levels; have increased fed circulatingC-peptide levels; have no change in fasted circulating C-peptide levels;have a decreased ratio of proinsulin to insulin (fed and fasted); haveno change in the ratio of C-peptide to insulin; have an increasedinsulin-positive area in the pancreas; have an increased islet numberper pancreas area; have increased beta-cell proliferation; and have anincreased number of insulin-producing beta cells. In addition, comparedto non-human animals without the mutation, non-human animals with amutated Slc30a8 locus can have increased fed plasma insulin levels afterblockade of the insulin receptor (e.g., using S961).

A mutated Slc30a8 locus disclosed herein can be a Slc30a8 locus encodinga truncated SLC30A8 protein (i.e., protein-truncating variants). Themutation causing the truncation can be, for example, a frameshiftmutation, a nonsense mutation, a splice site mutation, or an initiatorcodon SNV. See, e.g., Flannick et al. (2014) Nat. Genet. 46(4):357-363,herein incorporated by reference in its entirety for all purposes. Anonsense mutation is a mutation in which a sense codon that correspondsto one of the twenty amino acids specified by the genetic code ischanged to a chain-terminating codon (i.e., termination codon or stopcodon). A frameshift mutation arises when the normal sequence of codonsis disrupted by the insertion or deletion of one or more nucleotides,provided that the number of nucleotides added or removed is not amultiple of three. A frameshift mutation is a sequence change betweenthe translation initiation codon (start codon) and termination codon(stop codon) in which, compared to a reference sequence, translationshifts to another frame. For example, the reading frame can be shiftedone nucleotide in the 5′ direction (−1 frameshift) or one nucleotide inthe 3′ direction (+1 frameshift). A protein encoded by a gene with aframeshift mutation will be identical to the protein encoded by the wildtype gene from the N-terminus to the frameshift mutation, but differentbeyond that point. Such frameshifts can result in a prematuretermination codon. A splice site mutation can result, for example, inskipping of an exon and potentially a subsequent frameshift that resultsin a premature termination codon. In one example, a mutated Slc30a8locus disclosed herein can be a Slc30a8 locus in which the Slc30a8 genehas a premature termination codon (i.e., stop codon). In a specificexample, the mutation comprises, consists essentially of, or consists ofa nonsense mutation.

The mutation can be anywhere within the Slc30a8 locus. For example, themutation can be in the first exon, the second exon, the third exon, thefourth exon, the fifth exon, the sixth exon, the seventh exon, or theeighth exon of the Slc30a8 gene. Similarly, the mutation can be in aregion of the Slc30a8 gene corresponding to the first, second, third,fourth, fifth, sixth, seventh, or eighth exon of the human SLC30A8 genewhen optimally aligned with the human SLC30A8 gene. In a specificexample, the mutation is in the third exon of the Slc30a8 gene or in aregion of the Slc30a8 gene corresponding to the third exon of the humanSLC30A8 gene when optimally aligned with the human SLC30A8 gene. In amore specific example, the mutation is at the 3′ end of the third exonof the Slc30a8 gene or in a region of the Slc30a8 gene corresponding tothe 3′ end of third exon of the human SLC30A8 gene when optimallyaligned with the human SLC30A8 gene.

In one example, the mutation is at a residue corresponding to residue412 of the human SLC30A8 coding sequence (CDS) set forth in SEQ ID NO:21 (i.e., residue c.412, transcript accession number NM_173851) when thecoding sequence of the mutated Slc30a8 gene is optimally aligned withSEQ ID NO: 21. Alternatively, the mutation is at a residue within 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, or 500nucleotides of residue 412 of the human SLC30A8 coding sequence setforth in SEQ ID NO: 21 (i.e., residue c.412, transcript accession numberNM_173851) when the coding sequence of the mutated Slc30a8 gene isoptimally aligned with SEQ ID NO: 21.

In another example, the mutation is at a residue corresponding toresidue 409 of the mouse Slc30a8 coding sequence (CDS) set forth in SEQID NO: 20 (i.e., residue c.409, transcript accession number NM_173851.2)when the coding sequence of the mutated Slc30a8 gene is optimallyaligned with SEQ ID NO: 20. Alternatively, the mutation is at a residuewithin 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 100, 200, 300, 400, or500 nucleotides of residue 409 of the mouse Slc30a8 coding sequence setforth in SEQ ID NO: 20 (i.e., residue c.409, transcript accession numberNM_173851.2) when the coding sequence of the mutated Slc30a8 gene isoptimally aligned with SEQ ID NO: 20.

In another example, the mutation results in a stop codon in a codoncorresponding to residue 138 in the human SLC30A8 protein set forth inSEQ ID NO: 14 (i.e., amino acid 138 in UniProt accession numberQ8IWU4.2) when optimally aligned with SEQ ID NO: 14. Alternatively, thestop codon corresponds to a codon within 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, or 250 amino acids of residue 138 in thehuman SLC30A8 protein set forth in SEQ ID NO: 14 (i.e., amino acid 138in UniProt accession number Q8IWU4.2) when optimally aligned with SEQ IDNO: 14.

In another example, the mutation results in a stop codon in a codoncorresponding to residue 137 in the mouse SLC30A8 protein set forth inSEQ ID NO: 12 (i.e., amino acid 137 in UniProt accession numberQ8BGG0.1) when optimally aligned with SEQ ID NO: 12. Alternatively, thestop codon corresponds to a codon within 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, or 250 amino acids of residue 137 in themouse SLC30A8 protein set forth in SEQ ID NO: 12 (i.e., amino acid 137in UniProt accession number Q8BGG0.1) when optimally aligned with SEQ IDNO: 12.

The mutated Slc30a8 locus can be endogenous to the non-human animal. Ina specific example, the mutated Slc30a8 locus encodes an mRNA (cDNA)that comprises, consists essentially of, or consists of a sequence thatis at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQID NO: 18. The coding sequence of the mutated SLC30A8 locus cancomprise, consist essentially of, or consist of a sequence that is atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:22 or degenerates thereof encoding the same amino acid sequence. Theresulting SLC30A8 protein encoded by the mutated SLC30A8 locus cancomprise, consist essentially of, or consist of a sequence that is atleast 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:13.

Optionally, a mutated SLC30A8 locus can comprise other exogenous orheterologous elements. Examples of such elements can include selectioncassettes, reporter genes, recombinase recognition sites, or otherelements. As one example, a mutated SLC30A8 locus can comprise aremovable selection cassette (e.g., a self-deleting selection cassette)flanked by recombinase recognition sequences (e.g., loxP sites).Alternatively, the mutated SLC30A8 locus can lack other elements (e.g.,can lack a selection cassette and/or can lack a reporter gene). Examplesof suitable reporter genes and reporter proteins are disclosed elsewhereherein. Examples of suitable selection markers include neomycinphosphotransferase (neo_(r)), hygromycin B phosphotransferase (hyg_(r)),puromycin-N-acetyltransferase (puro_(r)), blasticidin S deaminase(bsr_(r)), xanthine/guanine phosphoribosyl transferase (gpt), and herpessimplex virus thymidine kinase (HSV-k). Examples of recombinases includeCre, Flp, and Dre recombinases. One example of a Cre recombinase gene isCrei, in which two exons encoding the Cre recombinase are separated byan intron to prevent its expression in a prokaryotic cell. Suchrecombinases can further comprise a nuclear localization signal tofacilitate localization to the nucleus (e.g., NLS-Crei). Recombinaserecognition sites include nucleotide sequences that are recognized by asite-specific recombinase and can serve as a substrate for arecombination event. Examples of recombinase recognition sites includeFRT, FRT11, FRT71, attp, att, rox, and lox sites such as loxP, lox511,lox2272, lox66, lox71, loxM2, and lox5171.

Other elements such as reporter genes or selection cassettes can beself-deleting cassettes flanked by recombinase recognition sites. See,e.g., U.S. Pat. No. 8,697,851 and US 2013/0312129, each of which isherein incorporated by reference in its entirety for all purposes. As anexample, the self-deleting cassette can comprise a Crei gene (comprisestwo exons encoding a Cre recombinase, which are separated by an intron)operably linked to a mouse Prm1 promoter and a neomycin resistance geneoperably linked to a human ubiquitin promoter. By employing the Prm1promoter, the self-deleting cassette can be deleted specifically in malegerm cells of F0 animals. The polynucleotide encoding the selectionmarker can be operably linked to a promoter active in a cell beingtargeted. Examples of promoters are described elsewhere herein. Asanother specific example, a self-deleting selection cassette cancomprise a hygromycin resistance gene coding sequence operably linked toone or more promoters (e.g., both human ubiquitin and EM7 promoters)followed by a polyadenylation signal, followed by a Crei coding sequenceoperably linked to one or more promoters (e.g., an mPrm1 promoter),followed by another polyadenylation signal, wherein the entire cassetteis flanked by loxP sites.

The mutated SLC30A8 locus can also be a conditional allele. For example,the conditional allele can be a multifunctional allele, as described inUS 2011/0104799, herein incorporated by reference in its entirety forall purposes. For example, the conditional allele can comprise: (a) anactuating sequence in sense orientation with respect to transcription ofa target gene; (b) a drug selection cassette (DSC) in sense or antisenseorientation; (c) a nucleotide sequence of interest (NSI) in antisenseorientation; and (d) a conditional by inversion module (COIN, whichutilizes an exon-splitting intron and an invertible gene-trap-likemodule) in reverse orientation. See, e.g., US 2011/0104799. Theconditional allele can further comprise recombinable units thatrecombine upon exposure to a first recombinase to form a conditionalallele that (i) lacks the actuating sequence and the DSC; and (ii)contains the NSI in sense orientation and the COIN in antisenseorientation. See, e.g., US 2011/0104799.

C. Non-Human Animal Genomes, Non-Human Animal Cells, and Non-HumanAnimals Comprising a Mutated Slc30a8 Locus

Non-human animal genomes, non-human animal cells, and non-human animalscomprising a mutated Slc30a8 locus as described elsewhere herein areprovided. The genomes, cells, or non-human animals can be male orfemale. The genomes, cells, or non-human animals can be heterozygous orhomozygous for the mutated Slc30a8 locus. A diploid organism has twoalleles at each genetic locus. Each pair of alleles represents thegenotype of a specific genetic locus. Genotypes are described ashomozygous if there are two identical alleles at a particular locus andas heterozygous if the two alleles differ.

The non-human animal genomes or cells provided herein can be, forexample, any non-human animal genome or cell comprising a Slc30a8 locusor a genomic locus homologous or orthologous to the human SLC30A8 locus.The genomes can be from or the cells can be eukaryotic cells, whichinclude, for example, animal cells, mammalian cells, non-human mammaliancells, and human cells. The term “animal” includes any member of theanimal kingdom, including, for example, mammals, fishes, reptiles,amphibians, birds, and worms. A mammalian cell can be, for example, anon-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or ahamster cell. Other non-human mammals include, for example, non-humanprimates, monkeys, apes, orangutans, cats, dogs, rabbits, horses,livestock (e.g., bovine species such as cows, steer, and so forth; ovinespecies such as sheep, goats, and so forth; and porcine species such aspigs and boars). Domesticated animals and agricultural animals are alsoincluded. The term “non-human” excludes humans.

The cells can also be any type of undifferentiated or differentiatedstate. For example, a cell can be a totipotent cell, a pluripotent cell(e.g., a human pluripotent cell or a non-human pluripotent cell such asa mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotentcell. Totipotent cells include undifferentiated cells that can give riseto any cell type, and pluripotent cells include undifferentiated cellsthat possess the ability to develop into more than one differentiatedcell types. Such pluripotent and/or totipotent cells can be, forexample, ES cells or ES-like cells, such as an induced pluripotent stem(iPS) cells. ES cells include embryo-derived totipotent or pluripotentcells that are capable of contributing to any tissue of the developingembryo upon introduction into an embryo. ES cells can be derived fromthe inner cell mass of a blastocyst and are capable of differentiatinginto cells of any of the three vertebrate germ layers (endoderm,ectoderm, and mesoderm).

The cells provided herein can also be germ cells (e.g., sperm oroocytes). The cells can be mitotically competent cells ormitotically-inactive cells, meiotically competent cells ormeiotically-inactive cells. Similarly, the cells can also be primarysomatic cells or cells that are not a primary somatic cell. Somaticcells include any cell that is not a gamete, germ cell, gametocyte, orundifferentiated stem cell. For example, the cells can be beta cells,pancreatic islet cells, or pancreatic cells.

Suitable cells provided herein also include primary cells. Primary cellsinclude cells or cultures of cells that have been isolated directly froman organism, organ, or tissue. Primary cells include cells that areneither transformed nor immortal. They include any cell obtained from anorganism, organ, or tissue which was not previously passed in tissueculture or has been previously passed in tissue culture but is incapableof being indefinitely passed in tissue culture. Such cells can beisolated by conventional techniques and include, for example, betacells.

Other suitable cells provided herein include immortalized cells.Immortalized cells include cells from a multicellular organism thatwould normally not proliferate indefinitely but, due to mutation oralteration, have evaded normal cellular senescence and instead can keepundergoing division. Such mutations or alterations can occur naturallyor be intentionally induced. Specific examples of immortalized celllines are the EndoC-βH2, 1.1B4, 1.4E7, 1.1E7, RIN, HIT, MIN, INS-1, andβTC cell lines. See, e.g., Scharfmann et al. (2014) J. Clin. Invest.124(5):2087-2098 and McCluskey et al. (2011) J. Biol. Chem.286(25):21982-21992, each of which is herein incorporated by referencein its entirety for all purposes. Numerous types of immortalized cellsare well known. Immortalized or primary cells include cells that aretypically used for culturing or for expressing recombinant genes orproteins.

The cells provided herein also include one-cell stage embryos (i.e.,fertilized oocytes or zygotes). Such one-cell stage embryos can be fromany genetic background (e.g., BALB/c, C57BL/6, 129, or a combinationthereof for mice), can be fresh or frozen, and can be derived fromnatural breeding or in vitro fertilization.

The cells provided herein can be normal, healthy cells, or can bediseased or mutant-bearing cells.

Non-human animals comprising a mutated Slc30a8 locus as described hereincan be made by the methods described elsewhere herein. The term “animal”includes any member of the animal kingdom, including, for example,mammals, fishes, reptiles, amphibians, birds, and worms. In a specificexample, the non-human animal is a non-human mammal. Non-human mammalsinclude, for example, non-human primates, monkeys, apes, orangutans,cats, dogs, horses, rabbits, rodents (e.g., mice, rats, hamsters, andguinea pigs), and livestock (e.g., bovine species such as cows andsteer; ovine species such as sheep and goats; and porcine species suchas pigs and boars). Domesticated animals and agricultural animals arealso included. The term “non-human animal” excludes humans. Preferrednon-human animals include, for example, rodents, such as mice and rats.

The non-human animals can be from any genetic background. For example,suitable mice can be from a 129 strain, a C57BL/6 strain, a mix of 129and C57BL/6, a BALB/c strain, or a Swiss Webster strain. Examples of 129strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, and 129T2. See, e.g., Festing et al. (1999)Mammalian Genome 10:836, herein incorporated by reference in itsentirety for all purposes. Examples of C57BL strains include C57BL/A,C57BL/An, C57BL/GrFa, C57BL/Kal_wN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. Suitablemice can also be from a mix of an aforementioned 129 strain and anaforementioned C57BL/6 strain (e.g., 50% 129 and 50% C57BL/6). Likewise,suitable mice can be from a mix of aforementioned 129 strains or a mixof aforementioned BL/6 strains (e.g., the 129S6 (129/SvEvTac) strain).

Similarly, rats can be from any rat strain, including, for example, anACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, aLEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer ratstrain such as Fisher F344 or Fisher F6. Rats can also be obtained froma strain derived from a mix of two or more strains recited above. Forexample, a suitable rat can be from a DA strain or an ACI strain. TheACI rat strain is characterized as having black agouti, with white bellyand feet and an RT1^(av1) haplotype. Such strains are available from avariety of sources including Harlan Laboratories. The Dark Agouti (DA)rat strain is characterized as having an agouti coat and an RT1^(av1)haplotype. Such rats are available from a variety of sources includingCharles River and Harlan Laboratories. Some suitable rats can be from aninbred rat strain. See, e.g., US 2014/0235933, herein incorporated byreference in its entirety for all purposes.

III. Methods of Making Non-Human Animals Comprising a Mutated Slc30a8Locus

Various methods are provided for making a non-human animal genome,non-human animal cell, or non-human animal comprising a mutated Slc30a8locus as disclosed elsewhere herein. Any convenient method or protocolfor producing a genetically modified organism is suitable for producingsuch a genetically modified non-human animal. See, e.g., Cho et al.(2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 andGama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each ofwhich is herein incorporated by reference in its entirety for allpurposes. Such genetically modified non-human animals can be generated,for example, through gene knock-in at a targeted Slc30a8 locus.

For example, the method of producing a non-human animal comprising amutated Slc30a8 locus can comprise: (1) modifying the genome of apluripotent cell to comprise the mutated Slc30a8 locus; (2) identifyingor selecting the genetically modified pluripotent cell comprising themutated Slc30a8 locus; (3) introducing the genetically modifiedpluripotent cell into a non-human animal host embryo; and (4) implantingand gestating the host embryo in a surrogate mother. For example, themethod of producing a non-human animal comprising a mutated Slc30a8locus can comprise: (1) modifying the genome of a pluripotent cell tocomprise the mutated Slc30a8 locus; (2) identifying or selecting thegenetically modified pluripotent cell comprising the mutated Slc30a8locus; (3) introducing the genetically modified pluripotent cell into anon-human animal host embryo; and (4) gestating the host embryo in asurrogate mother. Optionally, the host embryo comprising modifiedpluripotent cell (e.g., a non-human ES cell) can be incubated until theblastocyst stage before being implanted into and gestated in thesurrogate mother to produce an F0 non-human animal. The surrogate mothercan then produce an F0 generation non-human animal comprising themutated Slc30a8 locus.

The methods can further comprise identifying a cell or animal having amodified target genomic locus (i.e., a mutated Slc30a8 locus). Variousmethods can be used to identify cells and animals having a targetedgenetic modification.

The step of modifying the genome can, for example, utilize exogenousrepair templates (e.g., targeting vectors) to modify a Slc30a8 locus tocomprise an Slc30a8 mutation disclosed herein. As one example, thetargeting vector can be for generating a mutated Slc30a8 gene at anendogenous Slc30a8 locus (e.g., endogenous non-human animal Slc30a8locus), wherein the targeting vector comprises a 5′ homology armtargeting a 5′ target sequence at the endogenous Slc30a8 locus and a 3′homology arm targeting a 3′ target sequence at the endogenous Slc30a8locus, wherein the targeting vector comprises a mutation in the Slc30a8gene, wherein the mutated Slc30a8 gene encodes a truncated SLC30A8protein. As a specific example, the mutation can result in a stop codonin a codon corresponding to residue 138 in the human SLC30A8 protein.

The exogenous repair templates can be fornon-homologous-end-joining-mediated insertion or homologousrecombination. Exogenous repair templates can comprise deoxyribonucleicacid (DNA) or ribonucleic acid (RNA), they can be single-stranded ordouble-stranded, and they can be in linear or circular form. Forexample, a repair template can be a single-stranded oligodeoxynucleotide(ssODN).

Exogenous repair templates can also comprise nucleic acid insertsincluding segments of DNA to be integrated in the Slc30a8 locus.Integration of a nucleic acid insert in the Slc30a8 locus can result inaddition of a nucleic acid sequence of interest in the Slc30a8 locus,deletion of a nucleic acid sequence of interest in the Slc30a8 locus, orreplacement of a nucleic acid sequence of interest in the Slc30a8 locus(i.e., deletion and insertion).

Exogenous repair templates can also comprise a heterologous sequencethat is not present at an untargeted endogenous Slc30a8 locus. Forexample, an exogenous repair template can comprise a selection cassette,such as a selection cassette flanked by recombinase recognition sites.

Some exogenous repair templates comprise homology arms. If the exogenousrepair template acid also comprises a nucleic acid insert, the homologyarms can flank the nucleic acid insert. For ease of reference, thehomology arms are referred to herein as 5′ and 3′ (i.e., upstream anddownstream) homology arms. This terminology relates to the relativeposition of the homology arms to the nucleic acid insert within theexogenous repair template. The 5′ and 3′ homology arms correspond toregions within the Slc30a8 locus, which are referred to herein as “5′target sequence” and “3′ target sequence,” respectively.

A homology arm and a target sequence “correspond” or are “corresponding”to one another when the two regions share a sufficient level of sequenceidentity to one another to act as substrates for a homologousrecombination reaction. The term “homology” includes DNA sequences thatare either identical or share sequence identity to a correspondingsequence. The sequence identity between a given target sequence and thecorresponding homology arm found in the exogenous repair template can beany degree of sequence identity that allows for homologous recombinationto occur. For example, the amount of sequence identity shared by thehomology arm of the exogenous repair template (or a fragment thereof)and the target sequence (or a fragment thereof) can be at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity, such that the sequences undergo homologous recombination.Moreover, a corresponding region of homology between the homology armand the corresponding target sequence can be of any length that issufficient to promote homologous recombination. In some targetingvectors, the intended mutation in the Slc30a8 locus is included in oneof the homology arms. In other targeting vectors, the intended mutationin the Slc30a8 locus is included in an insert nucleic acid flanked bythe homology arms.

In cells other than one-cell stage embryos, the exogenous repairtemplate can be a “large targeting vector” or “LTVEC,” which includestargeting vectors that comprise homology arms that correspond to and arederived from nucleic acid sequences larger than those typically used byother approaches intended to perform homologous recombination in cells.LTVECs also include targeting vectors comprising nucleic acid insertshaving nucleic acid sequences larger than those typically used by otherapproaches intended to perform homologous recombination in cells. Forexample, LTVECs make possible the modification of large loci that cannotbe accommodated by traditional plasmid-based targeting vectors becauseof their size limitations. For example, the targeted locus can be (i.e.,the 5′ and 3′ homology arms can correspond to) a locus of the cell thatis not targetable using a conventional method or that can be targetedonly incorrectly or only with significantly low efficiency in theabsence of a nick or double-strand break induced by a nuclease agent(e.g., a Cas protein). LTVECs can be of any length and are typically atleast 10 kb in length. The sum total of the 5′ homology arm and the 3′homology arm in an LTVEC is typically at least 10 kb.

The screening step can comprise, for example, a quantitative assay forassessing modification of allele (MOA) of a parental chromosome. Forexample, the quantitative assay can be carried out via a quantitativePCR, such as a real-time PCR (qPCR). The real-time PCR can utilize afirst primer set that recognizes the target locus and a second primerset that recognizes a non-targeted reference locus. The primer set cancomprise a fluorescent probe that recognizes the amplified sequence.Modification-of-allele (MOA) assays including loss-of-allele (LOA) andgain-of-allele (GOA) assays are described, e.g., in US 2014/0178879 andFrendewey et al. (2010) Methods Enzymol. 476:295-307, each of which isherein incorporated by reference in its entirety for all purposes.Retention assays as described in US 2016/0145646 and WO 2016/081923,each of which is herein incorporated by reference in its entirety forall purposes, can also be used.

Other examples of suitable quantitative assays includefluorescence-mediated in situ hybridization (FISH), comparative genomichybridization, isothermic DNA amplification, quantitative hybridizationto an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beaconprobes, or ECLIPSE™ probe technology (see, e.g., US 2005/0144655,incorporated herein by reference in its entirety for all purposes).

An example of a suitable pluripotent cell is an embryonic stem (ES) cell(e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cellcan be generated, for example, through recombination by (a) introducinginto the cell one or more exogenous donor nucleic acids (e.g., targetingvectors) comprising an optional insert nucleic acid flanked, forexample, by 5′ and 3′ homology arms corresponding to 5′ and 3′ targetsites, wherein the insert nucleic acid or one of the homology armscomprises a mutated Slc30a8 locus or the mutation to be made at theSlc30a8 locus; and (b) identifying at least one cell comprising in itsgenome the insert nucleic acid integrated at the endogenous Slc30a8locus. The modified pluripotent cell can be generated, for example,through recombination by (a) introducing into the cell one or moretargeting vectors comprising an insert nucleic acid flanked by 5′ and 3′homology arms corresponding to 5′ and 3′ target sites, wherein theinsert nucleic acid comprises a mutated Slc30a8 locus or the mutation tobe made at the Slc30a8 locus; and (b) identifying at least one cellcomprising in its genome the insert nucleic acid integrated at theendogenous Slc30a8 locus. Any targeting vector can be used. In oneexample, a large targeting vector (LTVEC) is used. The LTVEC can be, forexample, at least 10 kb in length or can have 5′ and 3′ homology arms,the sum total of which is at least 10 kb in length. As another example,the targeting vector can be a single-stranded oligodeoxynucleotide(ssODN). The ssODN can be, for example, between about 80 to about 200nucleotides in length.

Alternatively, the modified pluripotent cell can be generated by (a)introducing into the cell: (i) a nuclease agent, wherein the nucleaseagent induces a nick or double-strand break at a recognition site withinthe endogenous Slc30a8 locus; and (ii) one or more exogenous donornucleic acids (e.g., targeting vectors) optionally comprising an insertnucleic acid flanked by, for example, 5′ and 3′ homology armscorresponding to 5′ and 3′ target sites located in sufficient proximityto the recognition site, wherein the insert nucleic acid or one of thehomology arms comprises the mutated Slc30a8 locus or the mutation to bemade at the Slc30a8 locus; and (c) identifying at least one cellcomprising a modification (e.g., integration of the insert nucleic acid)at the endogenous Slc30a8 locus. Alternatively, the modified pluripotentcell can be generated by (a) introducing into the cell: (i) a nucleaseagent, wherein the nuclease agent induces a nick or double-strand breakat a recognition site within the endogenous Slc30a8 locus; and (ii) oneor more targeting vectors comprising an insert nucleic acid flanked by5′ and 3′ homology arms corresponding to 5′ and 3′ target sites locatedin sufficient proximity to the recognition site, wherein the insertnucleic acid comprises the mutated Slc30a8 locus or the mutation to bemade at the Slc30a8 locus; and (c) identifying at least one cellcomprising a modification (e.g., integration of the insert nucleic acid)at the endogenous Slc30a8 locus. Any nuclease agent that induces a nickor double-strand break into a desired recognition site can be used.Examples of suitable nucleases include a Transcription Activator-LikeEffector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease,and Clustered Regularly Interspersed Short Palindromic Repeats(CRISPR)/CRISPR-associated (Cas) systems (e.g., CRISPR/Cas9 systems) orcomponents of such systems (e.g., CRISPR/Cas9). See, e.g., US2013/0309670 and US 2015/0159175, each of which is herein incorporatedby reference in its entirety for all purposes. Examples of suitablemethods for making targeted modifications to cells are described, e.g.,in US 2016/0145646 and WO 2016/081923, each of which is hereinincorporated by reference in its entirety for all purposes.

The donor cell can be introduced into a host embryo at any stage, suchas the blastocyst stage or the pre-morula stage (i.e., the 4-cell stageor the 8-cell stage). Progeny that are capable of transmitting thegenetic modification though the germline are generated. See, e.g., U.S.Pat. No. 7,294,754, herein incorporated by reference in its entirety forall purposes.

Alternatively, the method of producing the non-human animals describedelsewhere herein can comprise: (1) modifying the genome of a one-cellstage embryo to comprise the mutated Slc30a8 locus using the methodsdescribed above for modifying pluripotent cells; (2) selecting thegenetically modified embryo; and (3) implanting and gestating thegenetically modified embryo into a surrogate mother. Alternatively, themethod of producing the non-human animals described elsewhere herein cancomprise: (1) modifying the genome of a one-cell stage embryo tocomprise the mutated Slc30a8 locus using the methods described above formodifying pluripotent cells; (2) selecting the genetically modifiedembryo; and (3) gestating the genetically modified embryo in a surrogatemother. Progeny that are capable of transmitting the geneticmodification though the germline are generated.

Nuclear transfer techniques can also be used to generate the non-humananimals. Briefly, methods for nuclear transfer can include the steps of:(1) enucleating an oocyte or providing an enucleated oocyte; (2)isolating or providing a donor cell or nucleus to be combined with theenucleated oocyte; (3) inserting the cell or nucleus into the enucleatedoocyte to form a reconstituted cell; (4) implanting the reconstitutedcell into the womb of an animal to form an embryo; and (5) allowing theembryo to develop. In such methods, oocytes are generally retrieved fromdeceased animals, although they may be isolated also from eitheroviducts and/or ovaries of live animals. Oocytes can be matured in avariety of well-known media prior to enucleation. Enucleation of theoocyte can be performed in a number of well-known manners. Insertion ofthe donor cell or nucleus into the enucleated oocyte to form areconstituted cell can be by microinjection of a donor cell under thezona pellucida prior to fusion. Fusion may be induced by application ofa DC electrical pulse across the contact/fusion plane (electrofusion),by exposure of the cells to fusion-promoting chemicals, such aspolyethylene glycol, or by way of an inactivated virus, such as theSendai virus. A reconstituted cell can be activated by electrical and/ornon-electrical means before, during, and/or after fusion of the nucleardonor and recipient oocyte. Activation methods include electric pulses,chemically induced shock, penetration by sperm, increasing levels ofdivalent cations in the oocyte, and reducing phosphorylation of cellularproteins (as by way of kinase inhibitors) in the oocyte. The activatedreconstituted cells, or embryos, can be cultured in well-known media andthen transferred to the womb of an animal. See, e.g., US 2008/0092249,WO 1999/005266, US 2004/0177390, WO 2008/017234, and U.S. Pat. No.7,612,250, each of which is herein incorporated by reference in itsentirety for all purposes.

The various methods provided herein allow for the generation of agenetically modified non-human F0 animal wherein the cells of thegenetically modified F0 animal comprise the mutated Slc30a8 locus.Depending on the method used to generate the F0 animal, the number ofcells within the F0 animal that have the mutated Slc30a8 locus willvary. The introduction of the donor ES cells into a pre-morula stageembryo from a corresponding organism (e.g., an 8-cell stage mouseembryo) via for example, the VELOCIMOUSE® method allows for a greaterpercentage of the cell population of the F0 animal to comprise cellshaving the nucleotide sequence of interest comprising the targetedgenetic modification. For example, at least 50%, 60%, 65%, 70%, 75%,85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% of the cellular contribution of the non-human F0 animalcan comprise a cell population having the targeted modification.

The cells of the genetically modified F0 animal can be heterozygous forthe mutated Slc30a8 locus or can be homozygous for the mutated Slc30a8locus.

IV. Methods of Screening Compounds

The non-human animals having the mutated Slc30a8 loci described hereincan be used for screening compounds for activity potentially useful ininhibiting, ameliorating, or reducing type 2 diabetes or amelioratingtype-2-diabetes-like symptoms or screening compounds for activitypotentially harmful in promoting or exacerbating type 2 diabetes ortype-2-diabetes-like symptoms. Wild type non-human animals or non-humananimals that do not have the mutated Slc30a8 loci described herein canalso be used for screening compounds for activity potentially useful ininhibiting, ameliorating, or reducing type 2 diabetes or amelioratingtype-2-diabetes-like symptoms or screening compounds for activitypotentially harmful in promoting or exacerbating type 2 diabetes ortype-2-diabetes-like symptoms by monitoring phenotypic aspectsassociated with the protective mutated Slc30a8 loci described herein.Compounds having activity inhibiting, ameliorating, or reducing type 2diabetes or ameliorating type-2-diabetes-like symptoms are potentiallyuseful as therapeutics or prophylactics against type 2 diabetes.Compounds having activity promoting or exacerbating type 2 diabetes ortype-2-diabetes-like symptoms are identified as toxic and should beavoided as therapeutics or in other circumstances in which they may comeinto contact with humans (e.g., in foods, agriculture, construction, orwater supply).

Examples of compounds that can be screened include antibodies,antigen-binding proteins, site-specific DNA binding proteins (e.g.,CRISPR-Cas complexes), polypeptides, beta-turn mimetics,polysaccharides, phospholipids, hormones, prostaglandins, steroids,aromatic compounds, heterocyclic compounds, benzodiazepines, oligomericN-substituted glycines, and oligocarbamates. Large combinatoriallibraries of the compounds can be constructed by the encoded syntheticlibraries (ESL) method described in WO 1995/012608, WO 1993/006121, WO1994/008051, WO 1995/035503, and WO 1995/030642, each of which is hereinincorporated by reference in its entirety for all purposes. Peptidelibraries can also be generated by phage display methods. See, e.g.,U.S. Pat. No. 5,432,018, herein incorporated by reference in itsentirety for all purposes. Use of libraries of guide RNAs for targetingCRISPR-Cas systems to different genes are disclosed, e.g., in WO2014/204727, WO 2014/093701, WO 2015/065964, and WO 2016/011080, each ofwhich is herein incorporated by reference in its entirety for allpurposes.

Animal-based assays generally involve administering a compound to thenon-human animal comprising the mutated Slc30a8 locus and assessingsigns or symptoms closely resembling those of type 2 diabetes, signs orsymptoms related to type 2 diabetes, or phenotypic aspects related toglucose, insulin, glucose metabolism, or type 2 diabetes in humans forchange in response. The change can be assessed before and aftercontacting the non-human animal with the compound or by performing acontrol experiment performed with a control animal having the sameSlc30a8 mutation (e.g., wild type cohort sibling) without the compound.

Suitable phenotypic aspects that can be monitored include, for example,capacity to secrete insulin. For example, the capacity to secreteinsulin when fed a high-fat diet can be monitored. Alternatively, thecapacity to secrete insulin in response to hyperglycemia, such as severehyperglycemia, can be monitored. For example, the insulin secretion canbe in response to hyperglycemia induced by insulin receptor inhibition,such as hyperglycemia caused by the insulin receptor antagonist S961.

Other suitable phenotypic aspects that can be monitored includemitochondrial gene expression (e.g., expression of genes in themitochondrial oxidative phosphorylation (OXPHOS) system) or expressionof Hvcn1. Examples of such mitochondrial genes are described in moredetail in Example 2 and in Tables 6-8. Other suitable phenotypic aspectsthat can be monitored include ratio of insulin/C-peptide when fed acontrol chow diet or insulin clearance. Other suitable phenotypicaspects that can be monitored include islet zinc accumulation.

Other suitable phenotypic aspects that can be monitored include, forexample, (1) glucose-induced insulin secretion when fed a high-fat diet(e.g., circulating insulin levels after 20 weeks being fed on a high-fatdiet); (2) pancreatic beta-cell proliferation levels when fed a high-fatdiet (e.g., after 20 weeks being fed on a high-fat diet); (3) number ofpancreatic beta cells when fed a high-fat diet (e.g., after 20 weeksbeing fed on a high-fat diet); and (4) fed plasma insulin levels afterblockade of the insulin receptor (e.g., with using S961). Alternativelyor additionally, suitable phenotypic aspects that can be monitoredinclude, for example, proinsulin-to-insulin ratio when fed a high-fatdiet or insulin processing and health of pancreatic beta cells.

For example, activity for exacerbating type 2 diabetes can be identifiedby one or both of: (1) decreased capacity to secrete insulin (e.g., whenfed a high-fat diet or in response to hyperglycemia) or (2) reducedinsulin clearance (e.g., increased ratio of insulin/C-peptide when fed acontrol chow diet). Activity for exacerbating type 2 diabetes can alsobe identified by one or both of (1) increased mitochondrial geneexpression (e.g., expression of genes in the mitochondrial oxidativephosphorylation (OXPHOS) system) or (2) decreased expression of Hvcn1.

Activity for exacerbating type 2 diabetes can also be identified by oneor more or all of: (1) decreased glucose-induced insulin secretion whenfed a high-fat diet (e.g., decreased circulating insulin levels after 20weeks being fed on a high-fat diet); (2) decreased pancreatic beta-cellproliferation when fed a high-fat diet (e.g., after 20 weeks being fedon a high-fat diet); (3) decreased number of pancreatic beta cells whenfed a high-fat diet (e.g., after 20 weeks being fed on a high-fat diet);and (4) decreased fed plasma insulin levels after blockade of theinsulin receptor. Alternatively or additionally, activity forexacerbating type 2 diabetes can be identified by one or more or all ofincreased ratio of proinsulin-to-insulin and worsened insulin processingand health of pancreatic beta cells (e.g., as evidenced by increasedratio of proinsulin-to-insulin when fed a high-fat diet).

Alternatively, activity for ameliorating type 2 diabetes can beidentified by one or both of: (1) increased capacity to secrete insulin(e.g., when fed a high-fat diet or in response to hyperglycemia) or (2)increased insulin clearance (e.g., increased ratio of insulin/C-peptidewhen fed a control chow diet). Activity for exacerbating type 2 diabetescan also be identified by one or both of: (1) decreased mitochondrialgene expression (e.g., expression of genes in the mitochondrialoxidative phosphorylation (OXPHOS) system) or (2) increased expressionof Hvcn1.

Activity for ameliorating type 2 diabetes can also be identified by oneor more or all of (1) increased glucose-induced insulin secretion whenfed a high-fat diet (e.g., increased circulating insulin levels after 20weeks being fed on a high-fat diet); (2) increased pancreatic beta-cellproliferation when fed a high-fat diet (e.g., after 20 weeks being fedon a high-fat diet); (3) increased number of pancreatic beta cells whenfed a high-fat diet (e.g., after 20 weeks being fed on a high-fat diet);and (4) increased fed plasma insulin levels after blockade of theinsulin receptor. Alternatively or additionally, activity forameliorating type 2 diabetes can be identified by one or more or all ofdecreased ratio of proinsulin-to-insulin and improved insulin processingand health of pancreatic beta cells (e.g., as evidenced by decreasedratio of proinsulin-to-insulin when fed a high-fat diet).

Such phenotypic aspects can be assayed as described in the examplesprovided herein. The decrease or increase can be statisticallysignificant. For example, the decrease or increase can be by at leastabout 1%, at least about 2%, at least about 3%, at least about 4%, atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or 100%.

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise, if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleotide sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. When a nucleotide sequence encodingan amino acid sequence is provided, it is understood that codondegenerate variants thereof that encode the same amino acid sequence arealso provided. The amino acid sequences follow the standard conventionof beginning at the amino terminus of the sequence and proceedingforward (i.e., from left to right in each line) to the carboxy terminus.

TABLE 2 Description of Sequences. SEQ ID NO Type Description 1 DNA 8084AS Fwd Primer 2 DNA 8084 AS WT Probe 3 DNA 8084 AS Rev Primer 4 DNA 8084AS Mut Probe 5 DNA 8084 TD Fwd Primer 6 DNA 8084 TD Probe 7 DNA 8084 TDRev Primer 8 DNA Slc30a8 intron 2/Slc30a8 exon 3 pArg137*/ Slc30a8intron 3 9 DNA Slc30a8 intron 3/XhoI/(loxP) self-deleting neomycinselection cassette 10 DNA Self-deleting neomycin selection cassette(loxP)/ ICEUI/NheI/Slc30a8 intron 3 11 DNA Slc30a8 intron3/XhoI/loxP/ICEUI/NheI/Slc30a8 intron 3 12 Protein Mouse SLC30A8 WT(UniProt Q8BGG0.1) 13 Protein Mouse SLC30A8 p.Arg137X 14 Protein HumanSLC30A8 WT (UniProt Q8IWU4.2) 15 Protein Human SLC30A8 p.Arg138X 16 DNAMouse Slc30a8 cDNA WT (NM_172816.3) 17 DNA Human SLC30A8 cDNA WT(NM_173851.2) 18 DNA Mouse Slc30a8 cDNA c.409C > T 19 DNA Human SLC30A8cDNA c.412C > T 20 DNA Mouse Slc30a8 CDS WT 21 DNA Human SLC30A8 CDS WT22 DNA Mouse Slc30a8 CDS c.409C > T 23 DNA Human SLC30A8 CDS c.412C > T24 DNA Deleted Sequence in Mouse Slc30a8 in p.Arg137X Allele (MAID 8084)25 DNA Probe 26 DNA F Primer 27 DNA R Primer

EXAMPLES Example 1. Generation of Mice Comprising a Mutated Slc30a8Locus

To generate a mouse model carrying an allele corresponding to putativehuman SLC30A8 LOF allele (c.412C>T (transcript accession numberNM_173851.2), p.Arg138X), a mutated mouse Slc30a8 allele was generated(c.409C>T (transcript accession number NM_172816.3), p.Arg137X).Information on the mouse Slc30a8 and human SLC30A8 genes is provided inTable 3. An alignment of the human SLC30A8 and the mouse SLC30A8proteins is shown in FIG. 11. The mutated allele has a replacement of dCfor dT (c.409C>T) at the mouse Slc30a8 codon encoding R137 in exon 3,changing the codon from CGA to TGA (p.Arg137X), making a stop codonexpected to produce a truncated protein. The mutated allele also has aself-deleting neomycin selection cassette flanked by loxP sites(loxP-mPrm1-Crei-hUb1-em7-Neo-pA-loxP cassette (4,810 bp)) inserted atintron 3, deleting 29 bp of endogenous intron 3 sequence. The endogenousSlc30a8 locus is shown in FIG. 10A, and the targeted Slc30a8 locus isshown in FIG. 10B. The expected sequence for the region including theboundaries of Slc30a8 intron 2/Slc30a8 exon 3 pArg137*/Slc30a8 intron 3(denoted by horizontal line “A” in FIG. 10B) is set forth in SEQ ID NO:8. The expected sequence for the region including the boundaries ofSlc30a8 intron 3/XhoI/(loxP) self-deleting cassette (denoted byhorizontal line “B” in FIG. 10B) is set forth in SEQ ID NO: 9. Theexpected sequence for the region including the boundaries ofself-deleting cassette (loxP)/ICEUI/NheI/Slc30a8 intron 3 (denoted byhorizontal line “C” in FIG. 10B) is set forth in SEQ ID NO: 10.

TABLE 3 Mouse Slc30a8 and Human SLC30A8. Mouse Human Official SymbolSlc30a8 SLC30A8 NCBI GeneID 239436 169026 Primary Source MGI: 2442682HGNC: 20303 RefSeq mRNA NM_172816.3 NM_173851.2 ID UniProt ID Q8BGG0Q8IWU4 Genomic GRCm38.p4 GRCh38.p7 Assembly Location Chr 15: Chr 8:52295553-52335733 (+) 116950273-117176714 (+)

The targeted Slc30a8 locus after deletion of the self-deletingloxP-mPrm1-Crei-hUb1-em7-Neo-pA-loxP cassette is shown in FIG. 10C.After cassette deletion, loxP and cloning sites (77 bp) remain inSlc30a8 intron 3. The expected sequence for the region including theboundaries of Slc30a8 intron 2/Slc30a8 exon 3 pArg137*/Slc30a8 intron 3(denoted by horizontal line “A” in FIG. 10C) is set forth in SEQ ID NO:8. The expected sequence for the region including the boundaries ofSlc30a8 intron 3/XhoI/loxP/ICEUI/NheI/Slc30a8 intron 3 (denoted byhorizontal line “B” in FIG. 10C) is set forth in SEQ ID NO: 11.

A large targeting vector was generated comprising a 5′ homology armincluding 62 kb of sequence (but with the point mutation in the mouseSlc30a8 codon encoding R137) upstream from the region in Slc30a8targeted for deletion and 136 kb of the sequence downstream of theregion in Slc30a8 targeted for deletion. The large targeting vector wasdesigned to introduce a point mutation in the mouse Slc30a8 codon inexon 3 encoding R137 and to replace the 29 bp region in intron 3 withthe 4,810 bp self-deleting neomycin selection cassette. See FIG. 10B.The self-deleting neomycin selection cassette included the followingcomponents from 5′ to 3′: loxP site, mouse protamine (Prm1) promoter,Crei (Cre coding sequence optimized to include intron), human ubiquitinpromoter, EM7 promoter, neomycin phosphotransferase (neo_(r)) codingsequence, polyA, and loxP site. To generate the targeted allele,CRISPR/Cas9 components were introduced into F1H4 mouse embryonic stemcells together with the large targeting vector. Loss-of-allele assaysand allele-specific TAQMAN® assays using the primers and probes setforth in Table 4 were performed to confirm correct modification of theSlc30a8 allele. The loss-of-allele assays are denoted as “8084 TD”(TAQMAN® Downstream Assay) and the allele-specific assays are denoted as“8084 AS” (Allele-Specific Assay) in FIG. 10A. Such assays aredescribed, for example, in US 2014/0178879; US 2016/0145646; WO2016/081923; and Frendewey et al. (2010) Methods Enzymol. 476:295-307,each of which is herein incorporated by reference in its entirety forall purposes.

TABLE 4 Assays for Detection of Targeted Slc30a8 Locus. AssayPrimer or Probe Sequence 8084 AS WT Fwd CGAGGCCCCCTTCCAA (SEQ ID NO: 1)Probe (FAM-MGB) CATTTGGGTGGTATCGA (SEQ ID NO: 2) RevTGCGCAATCAGAGAATGTTACC (SEQ ID NO: 3) 8084 AS Mut Fwd CGAGGCCCCCTTCCAA(SEQ ID NO: 1) Probe (VIC-MGB) CATTTGGGTGGTATTGA (SEQ ID NO: 4) RevTGCGCAATCAGAGAATGTTACC (SEQ ID NO: 3) 8084 TD Fwd TGTGGGAATGTGAGCTCTCAAC(SEQ ID NO: 5) Probe (Cal O-BHQ1) CAGCTGACAGCAAGATTAATGGAAGTACC(SEQ ID NO: 6) Rev GACCCTGGAGACAGAAATGTC (SEQ ID NO: 7)

F0 mice were then generated from targeted ES cell clones using theVELOCIMOUSE® method. See, e.g., U.S. Pat. Nos. 7,576,259; 7,659,442;7,294,754; US 2008/007800; and Poueymirou et al. (2007) Nature Biotech.25(1):91-99, each of which is herein incorporated by reference in itsentirety for all purposes.

The sequence of the expected SLC30A8 protein expressed from the wildtype mouse locus is set forth in SEQ ID NO: 12. The sequence of theexpected truncated SLC30A8 protein expressed from the targeted mouselocus (SLC30A8 Arg137*) is set forth in SEQ ID NO: 13. An alignment ofthe two sequences is shown in FIG. 11.

Example 2. Characterization of SLC30A8 R138X Mice Summary

SLC30A8 encodes a zinc transporter that is primarily expressed in thepancreatic islets of Langerhans. In beta cells, it transports zinc intoinsulin-containing secretory granules. Loss-of-function (LOF) mutationsin SLC30A8 protect against type 2 diabetes in humans. Several Slc30a8knockout mouse lines have been characterized but do not fullyrecapitulate the human phenotype. Indeed, Slc30a8 deficient mice havereduced plasma insulin levels and impaired glucose tolerance, aphenotype that we replicate here in control Slc30a8-deficient mice. Wegenerated a knock-in mouse model carrying a mutation in the mouseSlc30a8 locus (c.409C>T (transcript accession number NM_172816.3),p.Arg137X) corresponding to the human SLC30A8 putative LOF mutation,R138X. Although the mutation in the mouse Slc30a8 gene is in the codonencoding amino acid residue 137 of the mouse SLC30A8 protein, these miceare referred to herein as “R138X mice” in reference to the correspondinghuman mutation. Interestingly, the R138X mice had increasedglucose-induced insulin secretion when fed a high-fat diet. This wasassociated with increased beta-cell proliferation and expansion of thebeta-cell area. Importantly, heterozygous R138X mice showed a similarincrease in glucose-induced insulin secretion. In contrast, chow-fedR138X mice had normal body weight, glucose tolerance, and pancreaticbeta-cell mass. Interestingly, in hyperglycemic conditions induced bythe insulin receptor antagonist S961, the R138X mice showed about 50%increase in insulin secretion. This effect was not associated withenhanced beta-cell proliferation or beta-cell mass. These data suggestthat the SLC30A8 R138X mutation in humans protects against type 2diabetes in part by increasing the capacity of beta cells to secreteinsulin. This may in part be mediated by an increased number ofpancreatic beta cells.

Introduction

SLC30A8 is a zinc transporter (ZnT8) located in the islet cells of theendocrine pancreas. In the beta cells, SLC30A8 transports zinc into theinsulin-containing granules. Two zinc ions bind with and stabilize ahexameric form of insulin triggering insulin crystallization. See, e.g.,Dodson and Steiner (1998) Curr. Opin. Struct. Biol. 8(2):189-194, hereinincorporated by reference in its entirety for all purposes. While lossof islet SLC30A8 substantially decreases islet zinc content and insulincrystallization, its effects on proinsulin processing, secretion andregulation of glucose metabolism are still unclear. See, e.g., Lemaireet al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106(35):14872-14877 andNicolson et al. (2009) Diabetes 58(9):2070-2083, each of which is hereinincorporated by reference in its entirety for all purposes. Severalgroups have generated Slc30a8 knockout (KO) mouse models either globallyor restricted to the pancreatic beta cells. The observed metabolicphenotype of these models is weak, and, although there are differencesbetween studies, most of the models show either no change or a mildimpairment of glucose tolerance associated with reduced or unchangedplasma insulin levels. See, e.g., Rutter et al. (2016) Proc. Nutr. Soc.75(1):61-72, herein incorporated by reference in its entirety for allpurposes. Most importantly, none of the studies have reported beneficialeffects associated with Slc30a8 deficiency. Therefore, it came as asurprise when Flannick and colleagues reported that haploinsufficiencyof SLC30A8 is protective against the development of type 2 diabetes(T2D) in humans. See, e.g., Flannick et al. (2014) Nat. Genet.46(4):357-363, herein incorporated by reference in its entirety for allpurposes. Their study identified 12 putative loss-of-function (LOF)variants that collectively decreased the risk for T2D by 65%. One of themost abundant variants, p.Arg138* (referred to as R138X herein), causinga premature stop codon, was individually associated with protection fromT2D and was reported to encode an unstable ZnT8 protein. See, e.g.,Flannick et al. (2014) Nat. Genet. 46(4):357-363, herein incorporated byreference in its entirety for all purposes. Thus, mouse and human dataseemingly contradict each other, and it is currently not clear how therare putative LOF variants reduce the risk of T2D.

To investigate this discrepancy between the effect of human SLC30A8putative LOF variants and the Slc30a8 KO mouse models as well as gaininsight as to why SLC30A8 putative LOF variants are protective inhumans, we generated and phenotyped a new Slc30a8 mouse model carryingthe R138X mutation as a representative human putative LOF variant.Interestingly, we found that the beta cells in the R138X mice have anextraordinary capacity to secrete insulin in response to severehyperglycemia.

Results

R138X Islets Appear to Show Loss of SLC30A8 Function Despite PartialProtection of the R138X Transcript from Nonsense-Mediated Decay.

To investigate how human SLC30A8 putative LOF variants influence therisk of T2D, we altered the codon of Slc30a8 in the mouse genomeencoding amino acid residue 137 (codon 137, equivalent to codon 138 inhuman) from CGA to TGA, changing the arginine into a stop codon (R138X)as detailed in Example 1. The R138X mice were born with the expectedMendelian ratio and no obvious growth abnormalities. RNA in situhybridization showed strong Slc30a8 mRNA staining in pancreatic isletsfrom wild type (WT) mice but no staining in islets from homozygousSlc30a8 KO mice, validating the specificity of the probe (FIG. 1A). Incontrast to islets from Slc30a8 KO mice, islets from R138X mice showed asubstantial amount of Slc30a8 RNA staining (FIG. 1A). Quantification ofSlc30a8 mRNA using real-time PCR revealed a 70% reduction in R138Xislets, although the absolute Slc30a8 transcript level in the R138Xislets (CT value: 22.2) was still high and comparable with theexpression level of the housekeeping gene Gapdh (CT value: 22.1) (FIG.1B). The presence of this high level of Slc30a8 RNA in the R138X micesuggests that the R138X transcript cannot effectively be degraded bynonsense-mediated mRNA decay, a process that degrades mRNAs carryingpremature translation termination codons. See, e.g., Holbrook et al.(2004) Nat. Genet. 36(8):801-808, herein incorporated by reference inits entirety for all purposes.

To determine whether the detected RNA in the R138X mice is translatedinto a truncated protein, we performed Western (immuno-) blotting forSLC30A8 protein using an antibody raised against an N-terminal peptideof the mouse protein. We were not able to identify a truncated proteinat the expected molecular weight (˜16 kDa for the monomer) in lysatesfrom isolated islets under the current conditions (FIG. 1C), despitebeing able to detect the R138X protein and stabilize it by proteasomalinhibition when overexpressed in HEK293 cells (FIG. 5). Similarly, massspectrometry analysis of whole cell lysates or immunoprecipitates fromWT and R138X islets did not identify SLC30A8 peptides in R138X islets(data not shown). In addition, zinc staining using dithizone revealedthat islets from KO and R138X mice were zinc depleted demonstrating theabsence of a functional SLC30A8 protein in both mouse lines (FIG. 1D).However, no significant change was observed in circulating zinc levels(FIG. 17). In summary, introduction of the human R138X putative LOFvariant into the mouse Slc30a8 gene appears to result in loss of SLC30A8function.

R138X Mice on Chow Diet have a Normal Metabolic Phenotype.

We first investigated whether the introduction of the R138X stop codonaltered metabolic parameters in chow-fed mice. WT and R138X mice did notdiffer in their body weight and circulating blood glucose and insulinlevels (FIGS. 2A-2C, 2L; Table 5). Because zinc has been implicated ininsulin processing and clearance, we also measured circulatingproinsulin and C-peptide levels and calculated the ratio of thesehormones to insulin (FIGS. 2C-2F). While there was no difference incirculating C-peptide, fasted proinsulin levels were slightly reduced inthe R138X mice (FIG. 2C). The ratio of proinsulin to insulin did notdiffer between WT and R138X mice, but the ratio of C-peptide to insulinin the fed state was slightly elevated suggesting reduced insulinclearance (FIGS. 2E-2F). Circulating proinsulin and C-peptide levelswere similar between WT and R138X mice (FIGS. 2C-2D). While the ratio ofproinsulin/C-peptide did not change, we did observe a mild decrease inthe ratio of insulin/C-peptide, suggesting higher insulin clearance inR138X mice similar to what has been observed in Slc30a8 KO mice (FIG.2P). See, e.g., Tamaki et al. (2013) J. Clin. Invest. 123(10):4513-4524,herein incorporated by reference in its entirety for all purposes.Reduced glucose tolerance was not apparent in the R138X mice, and we didnot observe changes in glucose-induced insulin secretion (FIGS. 2G-2H).Glucose tolerance and insulin sensitivity were similar in the R138X andcontrol mice, and we did not observe changes in glucose-induced insulinsecretion (FIGS. 2G, 2H, and 2M). In addition, the absence of islet zincdid not affect islet morphology as insulin and glucagon staining was notaltered (FIGS. 2I-2K; FIGS. 6A-6B). In addition, the absence of isletzinc did not affect islet morphology as beta-cell and alpha-cell masseswere not altered (FIGS. 2I, 2N, 6A, and 6E). Taken together, these datashow that the R138X variant does not affect glucose homeostasis orglucose-induced insulin secretion in chow-fed mice.

TABLE 5 Body Weight of SLC30A8 KO and R138X Mice. Genotype Sex Diet BW(g) WT Male Chow 26 ± 0.4 R138K Male Chow 27 ± 0.4 WT Male Chow 29 ± 1.1KO Male Chow 27 ± 0.6 WT Male HFD 55 ± 1.9 R138X Male HFD 57 ± 0.9 WTMale HFD 53 ± 1.4 KO Male HFD 54 ± 1.2 WT Male HFD 51 ± 0.9 R138 HETMale HFD 48 ± 0.8 WT Female HFD 54 ± 4.2 R138X Female HFD 54 ± 2.1

TABLE 6 Body Composition of Chow-Fed SLC30A8 KO and R138X Mice. Lean FatBone Bone Mineral Volume Volume Volume Bone Density Content Genotype Sex(cm³) (cm³) (cm³) (mgHA/cm³) (mgHA) WT Male 13.5 ± 0.4 2.6 ± 0.2 1.1 ±0.03 283 ± 3.3  320 ± 12.4 R138X Male 13.4 ± 0.3 3.1 ± 0.2 1.1 ± 0.11281 ± 2.8 311 ± 6.6 WT Female 13.7 ± 0.3 4.4 ± 0.5 1.2 ± 0.02 443 ± 3.4525 ± 5.4 R138X Female 13.8 ± 0.4 5.5 ± 0.5 1.2 ± 0.02 439 ± 5.2  519 ±15.8 WT Male 14.1 ± 0.3 4.4 ± 0.3 1.2 ± 0.01 294 ± 2.9 339 ± 6.3 KO Male13.9 ± 0.2 4.2 ± 0.4 1.2 ± 0.01 297 ± 2.9 343 ± 5.9

We also generated Slc30a8 KO mice in which the coding sequence from theSlc30a8 start codon to the Slc30a8 stop codon (i.e., the entire codingsequence) was deleted. We then phenotyped Slc30a8 KO mice on chow diet(FIGS. 7A-7K). In agreement with published results, the Slc30a8 KO miceshowed mild impaired glucose tolerance and reduced glucose-inducedplasma insulin levels (FIGS. 7B, 7G, 7H). See, e.g., Lemaire et al.(2009) Proc. Natl. Acad. Sci. U.S.A. 106(35):14872-14877; Nicolson etal. (2009) Diabetes 58(9):2070-2083; and Rutter et al. (2016) Proc.Nutr. Soc. 75(1):61-72, each of which is herein incorporated byreference in its entirety for all purposes. In addition, Slc30a8 KO micehad a higher ratio of C-peptide to insulin, suggesting increased insulinclearance similar to a recent report and presumably reflecting impairedzinc secretion from Slc30a8 KO islets. See, e.g., Tamaki et al. (2013)J. Clin. Invest. 123(10):4513-4524 and Mitchell et al. (2016) Mol.Endocrinol. 30(1):77-91, each of which is herein incorporated byreference in its entirety for all purposes.

Taken together, these data show that introduction of the R138X putativeLOF variant has minor effects on proinsulin processing and does notalter glucose homeostasis or glucose-induced insulin secretion in micemaintained on regular chow diet. This R138X phenotype thus differs fromthat observed in Slc30a8 KO mice, which have impaired glucose toleranceand reduced glucose-induced plasma insulin levels when maintained onregular chow diet.

R138X Mice on High-Fat Diet have Increased Insulin Secretion andBeta-Cell Number.

Since carriers of the human R138X are protected from development of T2D,we challenged the mice with a high-fat diet (HFD). Body weight and fedglucose levels did not differ between WT and R138X mice (Table 5, FIG.3A). However, after 20 weeks on HFD we observed higher circulatinginsulin levels in fed R138X mice compared to WT mice (FIG. 3B). Whilecirculating C-peptide levels mimicked the insulin levels, fastedproinsulin levels were lower in the R138X mice, resulting in a decreasedproinsulin to insulin ratio (FIGS. 3C-3F). Despite the increase in fedinsulin, glucose tolerance was not significantly improved in R138X mice(FIG. 3G). To examine insulin secretion in more detail, we measuredcirculating insulin after an oral glucose tolerance test (oGTT). InR138X mice, an oral glucose bolus doubled the amount of circulatinginsulin at 15 min when compared to WT mice (FIG. 3H). Strikingly, thiswas associated with an increased number of insulin-producing beta cells(FIG. 3I). Quantification of the insulin staining showed a doubling ofthe insulin-positive area, as well as the islet number (FIGS. 3J-3K). Incontrast, we detected no change in the number of alpha cells usingglucagon staining (FIGS. 6C-6D). Ki67 staining suggested that theincrease in beta-cell area was secondary to enhanced beta-cellproliferation. The number of insulin- and Ki67-double positive cellsincreased two-fold in the pancreas of R138X mice compared to WT mice. Asimilar, although less pronounced, metabolic phenotype was observed infemale R138X mice (FIGS. 8A-8K). After more than 20 weeks on HFD, femaleR138X mice showed a reduced proinsulin to insulin ratio when fasted, andan increase in circulating insulin levels associated with a highernumber of islets (FIGS. 8B-8K).

Because the protective phenotype of the R138X variant was observed inheterozygous carriers, we also analyzed heterozygous R138X mice on HFD(FIGS. 9A-9M). Western blot analysis showed a strong reduction ofSLC30A8 protein in islets from heterozygous R138X mice (FIG. 9A), whileislet zinc levels were comparable to WT mice when examined by dithizonestaining (FIG. 9B). Although the phenotype was milder compared to thehomozygous R138X mice, heterozygous R138X mice showed an increase inglucose-induced insulin secretion (FIG. 9J) associated with an expansionof beta-cell area after more than 20 weeks on HFD (FIG. 9K-9M). Takentogether, these data suggest that HFD mice carrying the human R138Xmutation have greater capacity to secrete insulin, which in part resultsfrom an increased number of beta cells.

As an alternative way to induce insulin resistance and increasebeta-cell insulin secretion, we used the insulin receptor antagonistS961 to block the insulin receptor in R138X mice and WT mice. Blockadeof the insulin receptor increased circulating glucose and insulin levelsas expected in both R138X mice and WT mice. However, we observed adoubling of the circulating insulin in R138X mice compared to WT mice.See FIG. 12. SLC30A8 KO mice do not show this increase in insulinsecretion over WT mice (data not shown). These data are consistent withthe data suggesting that HFD mice carrying the human R138X mutation havegreater capacity to secrete insulin.

Slc30a8 KO Mice on High-Fat Diet do not have Increased CirculatingInsulin Levels or Beta-Cell Number.

We also examined our Slc30a8 KO mice after 20 weeks on HFD (FIGS.4A-4K). These mice did not show an increase in fed circulating insulinlevels, but had reduced fasted insulin, proinsulin and C-peptide levels(FIGS. 4B-4D). Similar to the R138X mice, the proinsulin to insulinratio was lower in the Slc30a8 KO mice (FIG. 4E). However, the C-peptideto insulin ratio was elevated in the Slc30a8 KO mice similar to what wasobserved on chow diet. While glucose tolerance was unchanged, Slc30a8 KOmice showed lower glucose-induced circulating insulin levels. Incontrast to what we observed in the R138X mice, beta-cell number wasunchanged in the Slc30a8 KO mice (FIGS. 4I-4K).

R138X Mice have Increased Insulin Secretion in Response to InsulinReceptor Inhibition-Induced Hyperglycemia.

Next, we wanted to investigate a potential effect of the R138X mutationin metabolically challenged mice. We used the insulin receptorantagonist S961 to induce severe insulin resistance and hyperglycemia.See, e.g., Schaffer et al. (2008) Biochem. Biophys. Res. Commun.376(2):380-383, herein incorporated by reference in its entirety for allpurposes. As expected, S961 caused hyperglycemia (approximately 600mg/dl) in all treated animals, while the control animals kept normal fedglucose levels (approximately 200 mg/dl) (FIG. 13A). Circulating insulinlevels rose in response to S961 in all treated mice. Strikingly, R138Xmice had more than 50% (WT: 42.33±5.03 ng/ml; R138X: 67.75±19.16 ng/ml)higher circulating insulin levels upon S961-induced hyperglycemiacompared to S961-treated WT mice (FIG. 13B). The increase in plasmainsulin was not a result of improved proinsulin processing or decreasedinsulin clearance (FIGS. 14A-14E), or changes in circulating GLP-1levels between the R138X and WT mice (FIG. 13C). Circulating insulin washigher in fed and fasted condition in R138X mice treated with S961, butblood glucose levels did not differ between genotypes because of theinhibition of insulin action on the insulin receptor by S961 (FIGS.13D-13E). Of note, fasted insulin levels were substantially elevatedwith S961 treatment in WT and R138X mice when compared to PBS-treatedmice despite normal fasting blood glucose levels.

To investigate whether the higher insulin levels in the mutant mice werea consequence of increased beta-cell proliferation, we measuredKi-67/insulin positive beta cells. While beta-cell proliferation clearlyincreased with S961 treatment, there was no difference in beta-cellproliferation between R138X and WT mice (FIGS. 13F-13G). Consistent withthe proliferation data, there was no difference in islet or alpha-cellmass in S961-treated WT and R138X mice (FIGS. 13F and 13H, FIGS.14F-14G). Despite the large increase in blood insulin, beta cells fromS961-treated R138X mice did not appear to be more insulin-depleted thanbeta cells from the S961-treated WT mice (FIG. 13F). In summary, thesedata suggest that islets from the R138X mice have higher capacity tosecrete insulin when challenged with hyperglycemia.

Islets from R138X Mice have Decreased Mitochondrial Gene Expression andIncreased Hvcn1 Expression.

To understand the differences between WT and R138X islets in moredetail, we performed RNA sequencing on islets isolated from chow-fedmice. In confirmation with our RNAscope and TAQMAN® data (FIGS. 1A-1B),the amount of Slc30a8 mRNA was strongly reduced (from approximately 500RPKM in WT to approximately 100 RKPM in R138X), but not absent (FIG.15). We found that 61 genes (19 genes increased, 43 genes decreased)were differentially regulated in R138X islets (Table 7). Interestingly,this gene list does not contain genes involved in zinc metabolism orinsulin biosynthesis. However, the expression of insulin 2 (Ins2) wassignificantly down-regulated and the expression of insulin 1 (Ins1)trended lower too (FIG. 16A). This prompted us to look more carefullyinto the expression levels of beta-cell regulators, includingtranscription factors for insulin. As shown in FIG. 16B, Mafa wassignificantly upregulated in islets of R138X mice but had missed ourcut-off of 1.5-fold. Since Mafa positively regulates insulintranscription and its expression is usually positively associated withthe expression of the hormone, it does not explain the reduction theIns2 gene expression. Out of the 43 down-regulated genes, 12 genes weremitochondrial proteins and 7 belonged to the group of Oxphos genes(Table 7). We therefore looked into the expression level of all Oxphosgenes and noted that the expression of most of these genes was reduced(Table 8, Table 9). Complex I, III, and V were mainly affected with areduction in gene expression of about 40%, suggesting a reduced capacityto generate ATP.

TABLE 7 Differentially Regulated Genes in Islets from Chow-Fed WT andR138X Mice (Mitochondrial Genes Marked in Bold). Gene.ID Symbol Foldchange P value Description 170942 Erdr1 3.34 9.80E−04 erythroiddifferentiation regulator 1 74096 Hvcn1 2.87 3.90E−03 hydrogenvoltage-gated channel 1 12483 Cd22 2.79 2.70E−03 CD22 antigen 19354 Rac22.63 6.60E−04 RAS-related C3 botulinum substrate 2 12265 Ciita 2.472.50E−03 class II transactivator 17691 Sik1 2.37 7.10E−08 salt-induciblekinase 1 18636 Cfp 2.01 7.50E−03 complement factor properdin 17318 Mid11.79 1.70E−03 midline 1 170935 Grid2ip 1.75 8.60E−03 glutamate receptor,ionotropic, delta 2 (Grid2) interacting protein 1 666048 Trabd2b 1.724.20E−03 TraB domain-containing 2B 11899 Astn1 1.67 1.60E−03 astrotactin1 23984 Pde10a 1.66 4.50E−03 phosphodiesterase 10A 244723 Olfm2 1.627.40E−03 olfactomedin 2 17294 Mest 1.60 8.30E−04 mesoderm specifictranscript 319504 Nrcam 1.56 6.10E−06 neuron-glia-CAM-related celladhesion molecule 104601 Mycbpap 1.53 3.80E−03 MYCBP associated protein12545 Cdc7 1.53 8.00E−03 cell division cycle 7 (S. cerevisiae) 544617Arhgap27 1.52 3.50E−03 Rho GTPase activating protein 27 319760D130020L05Rik −1.52 1.10E−03 RIKEN cDNA D130020L05 gene 195531 Gm13152−1.54 3.40E−03 predicted gene 13152 66091 Ndufa3 −1.54 1.20E−03 NADHdehydrogenase (ubiquinone) 1 alpha subcomplex, 3 16334 Ins2 −1.547.20E−03 insulin II 235043 Tmem205 −1.54 8.70E−03 transmembrane protein205 14470 Rabac1 −1.56 2.90E−03 Rab acceptor 1 (prenylated) 14776 Gpx2−1.59 1.80E−07 glutathione peroxidase 2 66416 Ndufa7 −1.59 6.20E−03 NADHdehydrogenase (ubiquinone) 1 alpha subcomplex, 7 (B14.5a) 228715 Gm561−1.59 6.90E−03 predicted gene 561 66845 Mrpl33 −1.59 2.10E−03mitochondrial ribosomal protein L33 17178 Fxyd3 −1.59 3.10E−03 FXYDdomain-containing ion transport regulator 3 20832 Ssr4 −1.59 6.40E−03signal sequence receptor, delta 66594 Uqcr11 −1.61 7.50E−03ubiquinol-cytochrome c reductase, complex III subunit XI 66152 Uqcr10−1.61 7.30E−03 ubiquinol-cytochrome c reductase, complex III subunit X67267 Uqcc2 −1.61 7.60E−03 ubiquinol-cytochrome c reductase complexassembly factor 2 69038 Tmem258 −1.61 4.60E−03 transmembrane protein 25867885 1500011K16Rik −1.61 9.30E−03 RIKEN cDNA 1500011K16 gene 100502825Rpl37rt −1.61 5.40E−03 predicted gene 13826 66117 1110001J03Rik −1.614.40E−03 RIKEN cDNA 1110001J03 gene 69094 Tmem160 −1.61 8.50E−03transmembrane protein 160 67941 Rps27l −1.61 4.00E−03 ribosomal proteinS27-like 234421 Cib3 −1.64 5.30E−03 calcium and integrin binding familymember 3 11807 Apoa2 −1.67 3.80E−04 apolipoprotein A-II 20892 Stra13−1.67 2.90E−03 stimulated by retinoic acid 13 66477 Usmg5 −1.67 5.60E−03upregulated during skeletal muscle growth 5 68563 Dpm3 −1.67 2.50E−03dolichyl-phosphate mannosyltransferase polypeptide 3 22177 Tyrobp −1.679.90E−03 TYRO protein tyrosine kinase binding protein 69386 Hist1h4h−1.69 5.00E−03 histone cluster 1, H4h 268686 S100z −1.69 9.90E−04 S100calcium binding protein, zeta 73720 Cst6 −1.69 2.80E−03 cystatin E/M27425 Atp5l −1.69 4.90E−03 ATP synthase, H+ transporting, mitochondrialF0 complex, subunit G 68194 Ndufb4 −1.72 4.70E−03 NADH dehydrogenase(ubiquinone) 1 beta subcomplex 4 449000 Zfp960 −1.72 1.30E−03 zincfinger protein 960 93757 Immp2l −1.75 3.20E−03 IMP2 inner mitochondrialmembrane peptidase-like 

19735 Rgs2 −1.82 7.40E−03 regulator of G-protein signaling 2 17873Gadd45b −1.82 1.40E−04 growth arrest and DNA-damage-inducible 45 beta20335 Sec61g −1.82 3.20E−03 SEC61, gamma subunit 13190 Dct −1.894.10E−04 dopachrome tautomerase 11811 Apobec2 −1.89 3.30E−03apolipoprotein B mRNA editing enzyme, catalytic polypeptide 2 100169864SlcUbl4a −2.13 2.90E−05 Slc10a3-Ubl4 read-through 19782 Rmrp −2.178.40E−03 RNA component of mitochondrial RNase P 100568459 Bc1 −2.274.10E−03 brain cytoplasmic RNA 1 239436 Slc30a8 −6.67 1.40E−43 solutecarrier family 30 (zinc transporter), member 8

TABLE 8 Summary of Oxphos Genes Expression Changes in Islets of WT andR138X Mice. Complex Total Significantly Regulated (p < 0.05) % RegulatedComplex I 43 19 44.19 Complex II 4 1 25.00 Complex III 10 4 40.00Complex IV 19 5 26.32 Complex V 20 9 45.00

TABLE 9 Change in Expression of All Oxphos Genes in Isolated Islets fromWT and R138X Mice (Genes that Changed Significantly in Bold). R138X.vs.R138X.vs. WT_islets. WT_islets. Mitochondrial symbol fc pval Namecomplex MT-ND1 0.85 0.42 mitochondrially encoded NADH:ubiquinoneoxidoreductase core subunit 1 1 MT-ND2 0.93 0.67 mitochondrially encodedNADH:ubiquinone oxidoreductase core subunit 2 1 MT-ND3 0.81 0.27mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 3 1MT-ND4 0.84 0.36 mitochondrially encoded NADH:ubiquinone oxidoreductasecore subunit 4 1 MT-ND4L 0.8 0.23 mitochondrially encodedNADH:ubiquinone oxidoreductase core subunit 4L 1 MT-ND5 0.91 0.59mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 5 1MT-ND6 0.77 0.066 mitochondrially encoded NADH:ubiquinone oxidoreductasecore subunit 6 1 NDUFS1 0.95 0.54 NADH:ubiquinone oxidoreductase coresubunit S1 1 NDUFS2 0.79 0.0077 NADH:ubiquinone oxidoreductase coresubunit S2 1 NDUFS3 0.86 0.18 NADH:ubiquinone oxidoreductase coresubunit S3 1 NDUFS7 0.79 0.081 NADH:ubiquinone oxidoreductase coresubunit S7 1 NDUFS8 0.75 0.055 NADH:ubiquinone oxidoreductase coresubunit S8 1 NDUFV1 0.89 0.15 NADH:ubiquinone oxidoreductase coresubunit V1 1 NDUFV2 0.8 0.2 NADH:ubiquinone oxidoreductase core subunitV2 1 NDUFAB1 0.81 0.058 NADH dehydrogenase (ubiquinone) 1, alpha/betasubcomplex, 1 1 NDUFA1 0.68 0.033 NADH dehydrogenase (ubiquinone) 1alpha subcomplex, 1 1 NDUFA2 0.64 0.023 NADH dehydrogenase (ubiquinone)1 alpha subcomplex, 2 1 NDUFA3 0.65 0.0012 NADH dehydrogenase(ubiquinone) 1 alpha subcomplex, 3 1 NDUFA5 0.72 0.021 NADHdehydrogenase (ubiquinone) 1 alpha subcomplex, 5 1 NDUFA6 0.76 0.098NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6 (B14) 1 NDUFA70.63 0.0062 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7(B14.5a) 1 NDUFA8 0.91 0.35 NADH dehydrogenase (ubiquinone) 1 alphasubcomplex, 8 1 NDUFA9 0.87 0.22 NADH dehydrogenase (ubiquinone) 1 alphasubcomplex, 9 1 NDUFA10 0.91 0.15 NADH dehydrogenase (ubiquinone) 1alpha subcomplex 10 1 NDUFA11 0.71 0.016 NADH dehydrogenase (ubiquinone)1 alpha subcomplex 11 1 NDUFA12 0.69 0.017 NADH dehydrogenase(ubiquinone) 1 alpha subcomplex, 12 1 NDUFA13 0.7 0.01 NADHdehydrogenase (ubiquinone) 1 alpha subcomplex, 13 1 NDUFB2 0.55 0.012NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2 1 NDUFB3 0.82 0.17NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 1 NDUFB4 0.58 0.0047NADH dehydrogenase (ubiquinone) 1 beta subcomplex 4 1 NDUFB5 0.8 0.11NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5 1 NDUFB6 0.76 0.047NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 1 NDUFB7 0.79 0.12NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7 1 NDUFB8 0.72 0.022NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 1 NDUFB9 0.74 0.045NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9 1 NDUFB10 0.730.027 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10 1 NDUFB110.7 0.048 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11 1 NDUFC10.73 0.03 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1 1NDUFC2 0.75 0.074 NADH dehydrogenase (ubiquinone) 1, subcomplex unknown,2 1 NDUFS4 0.76 0.039 NADH dehydrogenase (ubiquinone) Fe — S protein 4 1NDUFS5 NaN NaN NADH dehydrogenase (ubiquinone) Fe—S protein 5 1 NDUFS60.72 0.017 NADH dehydrogenase (ubiquinone) Fe — S protein 6 1 NDUFV30.78 0.056 NADH dehydrogenase (ubiquinone) flavoprotein 3 1 SDHA 0.970.52 succinate dehydrogenase complex flavoprotein subunit A 2 SDHB 0.750.031 succinate dehydrogenase complex iron sulfur subunit B 2 SDHC 0.910.22 succinate dehydrogenase complex subunit C 2 SDHD 0.81 0.12succinate dehydrogenase complex subunit D 2 CYC1 0.91 0.37 cytochrome c13 MT-CYB 0.89 0.5 mitochondrially encoded cytochrome b 3 UQCRB 0.77 0.11ubiquinol-cytochrome c reductase binding protein 3 UQCRC1 0.93 0.48ubiquinol-cytochrome c reductase core protein 1 3 UQCRC2 0.83 0.12ubiquinol-cytochrome c reductase core protein 2 3 UQCRFS1 0.94 0.55ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 3UQCRH 0.64 0.016 ubiquinol-cytochrome c reductase hinge protein 3 UQCRQ0.66 0.021 ubiquinol-cytochrome c reductase complex III subunit VII 3UQCR10 0.62 0.0073 ubiquinol-cytochrome c reductase, complex III subunitX 3 UQCR11 0.62 0.0075 ubiquinol-cytochrome c reductase, complex IIIsubunit XI 3 COX4I1 0.73 0.058 cytochrome c oxidase subunit 4I1 4 C0X4I20.75 0.21 cytochrome c oxidase subunit 412 4 COX5A 0.91 0.37 cytochromec oxidase subunit 5A 4 COX5B 0.7 0.01 cytochrome c oxidase subunit 5B 4COX6A1 0.8 0.059 cytochrome c oxidase subunit 6A1 4 COX6A2 0.85 0.077cytochrome c oxidase subunit 6A2 4 COX6B1 0.71 0.011 cytochrome coxidase subunit 6B1 4 COX6B2 NaN NaN cytochrome c oxidase subunit 6B2 4COX6C 0.67 0.033 cytochrome c oxidase subunit 6C 4 COX7A1 NaN NaNcytochrome c oxidase subunit 7A1 4 COX7A2 0.62 0.017 cytochrome coxidase subunit 7A2 4 COX7B 0.74 0.072 cytochrome c oxidase subunit 7B 4COX7B2 NaN NaN cytochrome c oxidase subunit 7B2 4 COX7C 0.65 0.013cytochrome c oxidase subunit 7C 4 COX8A 0.84 0.12 cytochrome c oxidasesubunit 8A 4 COX8C NaN NaN cytochrome c oxidase subunit 8C 4 MT-CO1 0.870.45 mitochondrially encoded cytochrome c oxidase I 4 MT-CO2 0.9 0.55mitochondrially encoded cytochrome c oxidase II 4 MT-CO3 0.77 0.2mitochondrially encoded cytochrome c oxidase III 4 ATP5F1A 0.93 0.43 ATPsynthase F1 subunit alpha 5 ATP5F1B 0.88 0.11 ATP synthase F1 subunitbeta 5 ATP5F1C 0.82 0.13 ATP synthase F1 subunit gamma 5 ATP5F1D 0.850.16 ATP synthase F1 subunit delta 5 ATP5F1E 0.63 0.12 ATP synthase F1subunit epsilon 5 ATP5MC1 0.76 0.01 ATP synthase membrane subunit clocus 1 5 ATP5MC2 0.72 0.026 ATP synthase membrane subunit c locus 2 5ATP5MC3 0.87 0.19 ATP synthase membrane subunit c locus 3 5 ATP5MD 0.60.0056 ATP synthase membrane subunit DAPIT 5 ATP5ME 0.67 0.016 ATPsynthase membrane subunit e 5 ATP5MF 0.64 0.022 ATP synthase membranesubunit f 5 ATP5MG 0.59 0.0049 ATP synthase membrane subunit g 5 ATP5MPL0.68 0.03 ATP synthase membrane subunit 6.8PL 5 MT-ATP6 0.82 0.32mitochondrially encoded ATP synthase membrane subunit 6 5 MT-ATP8 0.820.36 mitochondrially encoded ATP synthase membrane subunit 8 5 ATP5PB0.84 0.18 ATP synthase peripheral stalk-membrane subunit b 5 ATP5PD 0.670.0081 ATP synthase peripheral stalk subunit d 5 ATP5PF 0.8 0.11 ATPsynthase peripheral stalk subunit F6 5 ATP5PO 0.78 0.046 ATP synthaseperipheral stalk subunit OSCP 5 ATP5IF1 0.74 0.07 ATP synthaseinhibitory factor subunit 1 5

Of note, the expression of the voltage-gated proton channel Hv1 (Hvcn1,alternate names VSOP, HV1) was upregulated in islets from R138X mice.Since this channel is Zn²⁺-regulated and present in the insulinsecretory granules, and loss of Hvcn1 expression impairs insulinsecretion in vivo and in vitro, this might be important for the observedphenotype in the R138X mice. See, e.g., Zhao et al. (2015) Biochem.Biophys. Res. Commun. 468(4):746-751; Wang et al. (2017) “TheVoltage-gated Proton Channel Hv1 Is Required for Insulin Secretion inPancreatic β Cells,” bioRxiv 097816, doi.org/10.1101/097816; and Qiu etal. (2016) Proc. Natl. Acad. Sci. U.S.A. 113(40):E5962-E5971, each ofwhich is herein incorporated by reference in its entirety for allpurposes.

Discussion

In this study, we generated a new Slc30a8 putative LOF mouse model(R138X mice) mimicking one of the two most common human SLC30A8 putativeLOFs (p.Arg138*). See, e.g., Flannick et al. (2014) Nat. Genet.46(4):357-363, herein incorporated by reference in its entirety for allpurposes. Data obtained from these mice can for the first time explainwhy SLC30A8 putative LOF mutations in humans are protective againstdevelopment of T2D. Introduction of the R138X mutation resulted in anincreased number of beta cells allowing for higher insulin secretion inan insulin resistant state. These data indicate that R138X beta cellscan therefore better compensate for the increased insulin demandpreventing and/or delaying the failure of beta cells and onset of T2D.Using this model, we show that the introduction of the R138X mutationresults in the secretion of 50% more insulin in response tohyperglycemia. These data suggest that the protection from T2D in humansis mediated, at least in part, by an increase in the insulin secretorycapacity allowing the beta cells to compensate for enhanced insulindemand thereby preventing or delaying the failure of these cells andonset of T2D. The present approach thus demonstrates the feasibility ofusing mouse genetics to explore the mechanisms of how identified humanT2D risk or protective genes act. The present approach also emphasizesthe utility of mouse genetics to explore the mechanisms of geneticvariants which affect the risk of T2D in humans.

More than eight different Slc30a8 knockout mouse models have beenreported. See, e.g., Lemaire et al. (2009) Proc. Natl. Acad. Sci. U.S.A.106(35):14872-14877; Nicolson et al. (2009) Diabetes 58(9):2070-2083;Tamaki et al. (2013) J. Clin. Invest. 123(10):4513-4524; Pound et al.(2009) Biochem. J. 421(3):371-376; Pound et al. (2012) PLoS One7(7):e40972; Wijesekara et al. (2010) Diabetologia 53(8):1656-1668; andHardy et al. (2012) Am. J. Physiol. Endocrinol. Metab.302(9):E1084-E1096, each of which is herein incorporated by reference inits entirety for all purposes. These models vary in genetic background,Slc30a8 targeting strategy, and the specific Cre-line that was used whenmice with beta-cell-specific deletions were generated. The metabolicphenotypes of these mice vary too. Differences in body weight gain,glucose tolerance and insulin secretion have been reported on chow andhigh-fat diet (HFD). See, e.g., Rutter et al. (2016) Proc. Nutr. Soc.75(1):61-72, herein incorporated by reference in its entirety for allpurposes. However, all published Slc30a8 knockout phenotypes pointtowards a worsening of glucose control with impaired glucose toleranceobserved in at least six publications. For comparative reasons, we alsogenerated a Slc30a8 KO mouse line on 100% C57BL/6 background, same asthe R138X. This knockout mouse phenocopied to a large extend thepublished data showing impaired glucose tolerance (on chow diet), lowerglucose-induced insulin secretion (on chow and HFD), and impairedinsulin clearance (on chow and HFD). On the contrary, we did not observea major metabolic phenotype, including worsening of glucose control inthe R138X mice on chow diet. The difference between the knockout and theR138X mouse lines became even more apparent after more than 20 weeks onHFD. Insulin secretion and the amount of beta cells was increased inR138X mice, but not in Slc30a8 KO mice. The late appearance of theobserved phenotype suggests that the compensatory increase in beta-cellproliferation and number happened rather late in HFD feeding. However,we cannot exclude the possibility that the number of beta cells differedalready after 10 weeks on HFD when no change in insulin secretion wasobserved. Hardy et al. observed an increase in the number of beta cellsand circulating insulin in their global Slc30a8 KO mouse after a HFDtreatment of 16 weeks. See, e.g., Hardy et al. (2012) Am. J. Physiol.Endocrinol. Metab. 302(9):E1084-E1096, herein incorporated by referencein its entirety for all purposes. However, this was most likely aconsequence of the deteriorated glucose control with marked obesity,hyperglycemia, and enhanced insulin resistance in their KO mice comparedto wild type mice, an effect not observed in the present study.Importantly, while Hardy et al. observed an increase in proinsulin toinsulin ratio in the Slc30a8 KO mice, we observed a decrease in thisratio in the R138X mice on HFD, suggesting that the beta cells hadimproved insulin processing and better health than the WT beta cells.See, e.g., Hardy et al. (2012) Am. J. Physiol. Endocrinol. Metab.302(9):E1084-E1096, herein incorporated by reference in its entirety forall purposes. Interestingly, the improvement in proinsulin to insulinratio was also observed in our Slc30a8 KO mice, showing that knockoutand R138X mice do not differ in all metabolic aspects. Since a highproinsulin to insulin ratio is associated with progression to T2D inhumans, this could also contribute to SLC30A8 LOF-mediated protectionfrom T2D. See, e.g., Kirchhoff et al. (2008) Diabetologia 51(4):597-601;Loopstra-Masters et al. (2011) Diabetologia 54(12):3047-3054; and Saadet al. (1990) J. Clin. Endocrinol. Metab. 70(5):1247-1253, each of whichis herein incorporated by reference in its entirety for all purposes.

All Slc30a8 KO phenotypes show either no change or a decrease incirculating insulin, which was in some instances associated withworsening of glucose tolerance. See, e.g., Nicolson et al., (2009)Diabetes 58(9):2070-2083; Pound et al. (2009) Biochem. J.421(3):371-376; and Mitchell et al. (2016) Mol. Endocrinol. 30(1):77-91,each of which is herein incorporated by reference in its entirety forall purposes. On the contrary, an increase in in vitro insulin secretionhas been observed in islets from three KO models. See, e.g., Nicolson etal., (2009) Diabetes 58(9):2070-2083; Tamaki et al. (2013) J. Clin.Invest. 123(10):4513-4524; and Hardy et al. (2012) Am. J. Physiol.Endocrinol. Metab. 302(9):E1084-E1096, each of which is hereinincorporated by reference in its entirety for all purposes. We did notobserve a major metabolic phenotype, including a change inglucose-induced insulin secretion, in chow-fed R138X mice. Whenchallenged with S961, however, these mice were able to secrete much moreinsulin than their WT counterparts. This increase in insulin secretionwas not secondary to an increase in beta-cell mass and has not beenreported in any Slc30a8 KO model.

It is unclear why the metabolic phenotype of the R138X mice differs somuch from the Slc30a8 KO mice, but the data suggest a potential gain offunction for the R138X allele. Currently, it is unclear whether theR138X mice are like Slc30a8 KO mice, since we cannot rule out that theremaining mRNA is translated into a truncated protein. We were not ableto detect a truncated version of the SLC30A8 protein that could readilyexplain phenotype differences in the islets of the R138X mice. However,the RNA is clearly present, and overexpressed R138X protein was detectedin HEK293 cells and accumulated by proteasomal inhibition (FIG. 5),which is contrary to what was previously observed in HeLa cells. See,e.g., Flannick et al. (2014) Nat. Genet. 46(4):357-363, hereinincorporated by reference in its entirety for all purposes. Thus, wecannot exclude the existence of low levels of protein, perhaps in asubset of the islet cells from the R138X mice. Independent of thepotential expression of SLC30A8 protein in R138X mice, dithizonestaining clearly indicated that the introduction of the R138X putativeLOF mutation resulted in a loss of islet zinc accumulation consistentwith what is described in the Slc30a8 KO mice. Another possibility isthat the truncated Slc30a8 mRNA, while no longer coding for a peptide,now serves a role as a long non-coding RNA, affecting the transcriptionof related genes (possibly including other Slc30a family members) intrans. See, e.g., Batista and Chang (2013) Cell 152(6):1298-1307, hereinincorporated by reference in its entirety for all purposes.

Changes in subcellular free Zn²⁺ may lead to increased proliferationand/or insulin secretion for a number of reasons. A plethora ofintracellular signaling systems are affected by free Zn²⁺ in mammaliancells with almost 10% of the cellular proteome able to bind these ions.See, e.g., Rutter et al. (2016) Proc. Nutr. Soc. 75(1):61-72, hereinincorporated by reference in its entirety for all purposes. Althoughfree Zn²⁺ concentrations are likely to be lowered globally in thecytosol by Slc30a8 inactivation in the beta cell, highly localizedincreases in free Zn²⁺ close to the plasma membrane in R138X beta cellsmight enhance signaling by receptor tyrosine kinases (e.g. insulin orIGF1 receptors) by inhibiting protein tyrosine phosphatases, thuspromoting cell growth. See, e.g., Gerber et al. (2014) Diabetologia57(8):1635-1644 and Bellomo et al. (2014) Metallomics 6(7):1229-1239,each of which is herein incorporated by reference in its entirety forall purposes. On the other hand, globally lowered cytosolic Zn²⁺ levelsare expected to reduce apoptotic rates, for example by interaction withcaspases. See, e.g., Fukamachi et al. (1998) Biochem. Biophys. Res.Commun. 246(2):364-369, herein incorporated by reference in its entiretyfor all purposes.

Insulin secretion may be upregulated in R138X mice through upregulationof the voltage-gated proton channel Hvcn1. Hvcn1 is present in secretorygranules and inhibited by zinc ions. See, e.g., Qiu et al. (2016) Proc.Nat. Acad. Sci. U.S.A. 113(40):E5962-E5971, herein incorporated byreference in its entirety for all purposes. In addition, Hvcn1 KO micehave decreased insulin secretion and impaired glucose control. Wang etal. (2017) “The Voltage-gated Proton Channel Hv1 Is Required for InsulinSecretion in Pancreatic β Cells,” bioRxiv 097816,doi.org/10.1101/097816, herein incorporated by reference in its entiretyfor all purposes. Since islets from the R138X mice are zinc-depleted,one would expect an increase in the activity of this channel. On top ofthat, we see a 3-fold upregulation of the Hvcn1 transcript in isletsfrom R138X mice. While these data suggest that the increase in insulinsecretion is at least in part mediated by Hvcn1, further experimentshave to validate this hypothesis. Another interesting finding is thereduction of mitochondrial gene expression in R138X mice. This seemsrather counterintuitive, since ATP synthesis is required for properinsulin secretion. See, e.g., Wiederkehr and Wollheim (2012) Mol. Cell.Endocrinol. 353(1-2):128-137, herein incorporated by reference in itsentirety for all purposes. However, a recent study described analternative pathway to activate insulin secretion in beta cells. See,e.g., De Marchi et al. (2017) J. Cell. Sci. 130(11):1929-1939, hereinincorporated by reference in its entirety for all purposes. This pathwayis independent of ATP synthesis (oligomycin-resistant), depends onpermissive cytosolic Ca²⁺, is accompanied by smaller mitochondrialglutathione reduction, and is achieved by activating mitochondrialcomplex II. This is interesting taking into consideration that Gpx2expression (glutathione peroxidase 2) (Table 7) and only one gene fromOxphos Complex II was reduced in the R138X mice. Correspondingly,deletion of the protein kinase Liver Kinase B1 (LKB1) selectively inbeta cells leads to marked mitochondrial defects and impaired Ca²⁺dynamics, while glucose-stimulated insulin secretion is enhanced mostlikely due to the upregulation of the latter pathway. See, e.g., Swisaet al. (2015) J. Biol. Chem. 290(34):20934-20946, herein incorporated byreference in its entirety for all purposes. Further experimentsexamining respiration and insulin secretion can be undertaken toinvestigate whether this alternative pathway is predominantly used inbeta cells from R138X mice.

Restoration of beta-cell number is a key strategy for the development ofnovel T2D drugs. One of the biggest challenges to this approach is thatfactors that promote beta-cell replication in rodents have often failedto do so in human beta cells. However, our data in mice modeling thehuman SLC30A8 R138X mutation suggest that an increase in beta-cellnumber is one of the mechanisms underlying the T2D protective effects ofsome SLC30A8 mutations in humans. Therefore, understanding themechanisms underlying the observed expansion of beta-cell area in R138Xmice could lead to the identification of novel therapeutic targets withhigher chance of translating into humans.

In conclusion, our data from the R138X mice offer for the first time anexplanation as to why humans carrying the R138X mutation are protectedagainst T2D. See, e.g., Flannick et al. (2014) Nat. Genet.46(4):357-363, herein incorporated by reference in its entirety for allpurposes. It therefore is a relevant model to study the mechanismunderlying this protection. This is especially important given thatincreasing insulin secretion is a prominent therapeutic strategy tocombat T2D. Therefore, understanding the mechanism underlying theincreased insulin secretion in R138X mice could lead to theidentification of novel therapeutic targets.

Mechanism of Action

We have generated a new mouse model (R138X) mimicking the human SLC30A8LoF mutation p.Arg138* that protects humans from the development of type2 diabetes. R138X mice in high-fed-diet conditions have increasedinsulin secretion associated with increased beta-cell mass andproliferation. Alternatively, when we induce hyperglycemia using S961(insulin receptor inhibitor), R138X mice have 50% increased insulinsecretion that is independent from beta-cell mass and proliferationchanges compared to WT mice. This suggests that the insulin receptor isrequired for the increase in proliferation in R138X mice. In addition,the insulin receptor in beta cells has been shown to be required for anincrease in beta-cell proliferation in mice on high-fat diet. SinceR138X mice have increased capacity to secrete insulin, the higherinsulin can act on the insulin receptor in beta cells and induceproliferation to a larger extent than in WT mice. To investigate this,activation of the insulin pathway is examined by doing histology forp-Akt, p-S6, and p-Erk in pancreata from WT and R138X mice treated withhigh-fat diet or S961. In addition, isolated islets are examined todetermine whether they have higher p-AKT and p-Erk levels and whetherthey proliferate more in vitro.

Methods

Animals.

All procedures were conducted in compliance with protocols approved bythe Regeneron Pharmaceuticals Institutional Animal Care and UseCommittee. The Slc30a8 R138X and knockout mouse lines were made in pureC57BL/6NTac background using VELOCIGENE technology. See, e.g.,Valenzuela et al. (2003) Nat. Biotechnol. 21(6):652-659, hereinincorporated by reference in its entirety for all purposes. For thegeneration of the knockout mouse line, the whole Slc30a8 coding regionwas deleted and replaced with a LacZ gene. The neomycin gene was removedthrough the use of a self-deleting neomycin selection cassette. For thegeneration of the R138X mice, nucleotide 409 was changed from T into Cin exon 3, which changes the arginine into a stop codon. The mutatedallele has a self-deleting neomycin selection cassette flanked by loxPsites inserted at intron 3, deleting 29 bp of endogenous intron 3sequence (CAGCTGACAGCAAGATTAATGGAAGTACC (SEQ ID NO: 24)). Mice werehoused (up to five mice per cage) in a controlled environment (12-hlight/dark cycle, 22±1° C., 60-70% humidity) and fed ad libitum witheither chow (Purina Laboratory 23 Rodent Diet 5001, LabDiet) or high-fatdiet (Research Diets, D12492; 60% fat by calories) starting at age 12-18weeks. All data shown are compared to WT littermates.

Glucose Tolerance Test.

Mice were fasted overnight (16 hr) followed by oral gavage of glucose(Sigma) at 2 g/kg body weight. Blood samples were obtained from the tailvein at the indicated times and glucose levels were measured using theAlphaTrak2 glucometer (Abbott). Submandibular bleeds for insulin weredone at 0, 15, and 30 min post-injection in separate experiments to notinterfere with glucose measurements.

Insulin Tolerance Test.

Mice were fasted for 4 hr followed by intraperitoneal injection ofeither 0.75 U/kg (chow-fed) or 1.5 U/kg (high-fat-fed) of human insulin(Eli Lilly). Blood samples were obtained from the tail vein at theindicated times and glucose was measured using the AlphaTrak2 glucometer(Abbott).

Plasma Insulin, Proinsulin and C-peptide Measurements.

Submandibular bleeds of either overnight fasted or fed animals were donein the morning or following an oGTT. Insulin was analyzed with the mouseinsulin ELISA (Mercodia). Proinsulin was analyzed with the mouseproinsulin ELISA (Mercodia). C-peptide was analyzed with the mouseC-peptide ELISA (ALPCO). All ELISAs were performed according to themanufacturer's instructions.

Histology.

Pancreata were fixed in 10% neutral buffered formalin solution for 48 hand then embedded in paraffin. Two sections of the pancreas from eachanimal were stained with an α-glucagon (REGN745, an α-glucagonmonoclonal antibody generated in-house) or an α-insulin (Dako) antibody,and areas of glucagon and insulin positive cells were measured usingHalo digital imaging analysis software (Indica Labs). The percent ofglucagon and insulin positive areas in proportion to the whole pancreasarea were calculated. The islet number per slide was counted andnormalized to the whole pancreas area. The analysis was set to captureevery cluster of insulin-positive cells down to a single cellresolution. We excluded artifacts (e.g., debris) smaller than one singlebeta cell (<150 μm²; area calculated using a diameter of about 14 μm).For fluorescence staining, pancreas sections were stained with acombination of α-insulin (Dako, A0564) antibody and α-KI67 antibody(R&D) followed by appropriate secondary antibodies. Fluorescent signalwas detected using a microscope slide scanner (Zeiss Axio Scan.Z1).Islet cell types were quantified using the HALO image analysis using theCytonuclear Fluorescence module (Indica Labs). The percent of glucagon-and insulin-positive areas relative to the whole pancreas area werecalculated. Taking the area as basis, α-cell mass was calculated bymultiplying the α-cell area for each animal by the weight of theanimal's pancreas. To quantify islet mass, islet number and size wasmeasured by counting the number of insulin-positive islets on a section.Every stained area larger than 150 μm was counted as an islet. The isletnumber was normalized to the whole pancreas area multiplied by theindividual pancreas weight to get the islet mass.

RNA In Situ Hybridization.

For RNA analysis, pancreas tissue was permeabilized and hybridized withcombinations of mRNA probes for mouse Gcg, Ins2, and Slc30A8 accordingto the manufacturer's instructions (Advanced Cell Diagnostics). Afluorescent kit was used to amplify the mRNA signal. Fluorescent signalwas detected using a microscope slide scanner (Zeiss Axio Scan.Z1).

Islet Isolation and Dithizone Staining.

Mouse islets were isolated by density gradient separation afterperfusing pancreas with Liberase TL (Roche, #05401020001) through thecommon bile duct. Following 13 min digestion at 37° C., the pancreassolution was washed and filtered through a 400-μm wire mesh strainer andislets were separated by Histopaque gradient centrifugation(Sigma-Aldrich, #H 1077) or by hand picking under a dissectionmicroscope. Isolated islets were cultured overnight in RPMI-1640 medium(Gibco), supplemented with 10% (v/v) FBS, 10 mM HEPES, 50 μMβ-mercaptoethanol, 1.0 mM sodium-pyruvate, 100 U/mL penicillin and 100μg/mL streptomycin at 37° C. with a 5% CO₂ in air atmosphere. Up to 50islets were hand-picked and transferred to a new dish containing 100g/ml dithizone solution. Islets were stained for up to 10 min andtransferred back into a PBS solution for microscopy (Zeiss Confocalmicroscope).

Western (Immuno-) Blot Analysis.

Islets were lysed in RIPA lysis buffer and the protein concentration wasdetermined. 20 μg of total cell lysate was resolved by SDS/PAGE usingCriterion TGX 4-20% precast gel (Bio-Rad) under reducing conditions andtransferred to nitrocellulose membranes. The membranes were probed witha polyclonal α-mouse-SLC30A8 Rabbit Ab (custom made from Thermo Fisher)and detected using an enhanced chemiluminescent detection system. TheSLC30A8 polyclonal Ab was raised against a peptide spanning amino acids29-43 of the mouse SLC30A8 protein.

R138X Overexpression and Proteasomal Inhibition.

Human SLC30A8 WT and the first 137 amino acids followed by a stop codonwere cloned with a C-terminal myc tag. HEK293 cells (0.4×10⁶ cells/well)were seeded into a 6-well plate. The next day, cells were transfectedwith 0.1 μg DNA/well using Mirus TransIT-TL1 following themanufacturer's instructions. Two days after transfection either DMSO,proteasomal (10 μM MG-132 or 1 μM Epoxomicin) or lysosomal inhibitors(10 μg/ml Cloroquine Salt) were spiked in. Cells were harvested after 6h treatment in RIPA buffer. 30 μg of the cell lysates were analyzed byWestern blotting.

qPCR Analysis.

Between 37.5 nanograms and 375 nanograms of RNA, per RT-qPCR assay to berun, was mixed with SuperScript® VILO™ Master Mix (ThermoFisher,Cat#11755500) and cycled according to the manufacturer's instructions.The cDNA was diluted with nuclease free water to between 0.5 nanogramsper microliter and 5 nanograms per microliter. RT-qPCR assays were madeby combining water, mastermix (ThermoFisher, Cat #4370074 or Bioline,Cat #CSA-01113), and 20× assay mix. The assay mix was a commerciallyavailable mixture of forward and reverse primers combined with afluorescently labeled and quenched probe sequence (ThermoFisher,Cat#351372 or LGC BioSearch, Cat# DLO-RFBL-MIX). Samples were run intriplicate on a 384-well plate (ThermoFisher, Cat#4343370) by pipetting5 microliters diluted cDNA and 10 microliters of appropriate RT-PCRassay into each well. The 384-well plate was covered with an opticallyclear seal (Agilent, Cat#16985-001), spun down, and read on an ABI7900HT Fast Real-Time PCR System with 384-Well Block Module andAutomation Accessory (ThermoFisher, Cat#4329002) for 40 cycles accordingto the specifics of the mastermix used. The sequences of the Slc30a8probe and primers are as follows:

Probe: (SEQ ID NO: 25) TCCAAAACTGGGCAGTGAGTTCAACA, Forward primer:(SEQ ID NO: 26) AATTGCAGTGCTGCTTTGC, Reverse primer: (SEQ ID NO: 27)AGCTGCGGCTGTTGTTGTC.

RNA Preparation.

Islets were isolated as described above and incubated over night at 37°C. The next day, 500 islets per genotype were picked into Trizol(Invitrogen) as one sample and kept at −80° C. until RNA extraction andsequencing. Five different WT samples and four different R138× sampleswere analyzed. Total RNA was purified from all samples using MagMAX™-96for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies)according to the manufacturer's specifications. Genomic DNA was removedusing MagMAX™Turbo™DNase Buffer and TURBO DNase from the MagMAX kitlisted above (Ambion by Life Technologies). mRNA was purified from totalRNA using Dynabeads® mRNA Purification Kit (Invitrogen). Strand-specificRNA-seq libraries were prepared using KAPA mRNA-Seq Library PreparationKit (Kapa Biosystems). Twelve-cycle PCR was performed to amplifylibraries. Sequencing was performed on Illumina HiSeq®2500 (Illumina) bymultiplexed single-read run with 33 cycles.

RNAseq Read Mapping and Statistical Analysis of Differentially ExpressedRNA.

Raw sequence data (BCL files) were converted to FASTQ format viaIllumina bcl2fastq v2.17. Reads were decoded based on their barcodes andread quality was evaluated with FastQC. Reads were mapped to the mousegenome (NCBI GRCm38) using ArrayStudio® software (OmicSoft®) allowingtwo mismatches. Reads mapped to the exons of a gene were summed at thegene level. Differential expressed genes were identified by DESeq2package and significantly perturbed genes were defined with fold changesno less than 1.5 in either up or down direction and with p-values of atleast 0.01.

Data Analyses.

Data are reported as mean±SEM. Statistical analyses were performed usingPrism 6.0 (GraphPad Software). All parameters were analyzed by two-wayANOVA (*p) and Students TTest (^(#)p) as indicated always comparinggenotypes not treatments; a threshold of P<0.05 was consideredstatistically significant. Significant difference between genotypes bytwo-way ANOVA is indicated on the top of the graph. Significantdifferences of specific data points are indicated on top of thepoint/graph. *p/^(#)p<0.5, **p/^(##)p<0.01, ***p/^(###)p<0.001,****p/^(####)p<0.0001.

1. A non-human animal whose genome comprises an endogenous Slc30a8 locuscomprising a mutated Slc30a8 gene, wherein the mutated Slc30a8 geneencodes a truncated SLC30A8 protein and results in the non-human animalhaving an enhanced capacity for insulin secretion relative to anon-human animal without the mutation.
 2. The non-human animal of claim1, wherein the enhanced capacity for insulin secretion is in response tohyperglycemia.
 3. The non-human animal of claim 2, wherein the non-humananimal has increased insulin secretion in response to hyperglycemiainduced by insulin receptor inhibition relative to the non-human animalwithout the mutation.
 4. The non-human animal of claim 3, wherein theincreased insulin secretion in response to hyperglycemia induced byinsulin receptor inhibition is not associated with increased beta-cellproliferation or beta-cell mass relative to the non-human animal withoutthe mutation.
 5. The non-human animal of claim 1, wherein the enhancedcapacity for insulin secretion is when fed a high-fat diet.
 6. Thenon-human animal of claim 5, wherein the non-human animal has increasedinsulin secretion relative to the non-human animal without the mutationwhen fed a high-fat diet, wherein the increased insulin secretion isassociated with increased beta-cell proliferation or beta-cell massrelative to the non-human animal without the mutation.
 7. The non-humananimal of claim 6, wherein the increased beta-cell proliferation isinsulin-receptor-dependent.
 8. The non-human animal of claim 1, whereinthe mutated Slc30a8 gene has a premature termination codon.
 9. Thenon-human animal of claim 1, wherein the mutated Slc30a8 gene comprisesa mutation is in the third exon of the Slc30a8 gene.
 10. The non-humananimal of claim 9, wherein the mutation is at the 3′ end of the thirdexon of the Slc30a8 gene.
 11. The non-human animal of claim 1, whereinthe mutated Slc30a8 gene comprises a nonsense mutation.
 12. Thenon-human animal of claim 11, wherein the nonsense mutation is in acodon corresponding to the codon encoding R138 in SEQ ID NO: 14 when theSLC30A8 protein encoded by the mutated Slc30a8 gene is optimally alignedwith SEQ ID NO:
 14. 13. The non-human animal of claim 11, wherein thenonsense mutation is at a position corresponding to residue 412 in SEQID NO: 21 when the coding sequence of the mutated Slc30a8 gene isoptimally aligned with SEQ ID NO:
 21. 14. The non-human animal of claim1, wherein the mutated Slc30a8 gene is endogenous to the non-humananimal.
 15. The non-human animal of claim 1, wherein the non-humananimal is a rat or a mouse.
 16. The non-human animal of claim 15,wherein the non-human animal is a mouse.
 17. The non-human animal ofclaim 16, wherein the mutated Slc30a8 gene encodes a SLC30A8 proteincomprising the sequence set forth in SEQ ID NO:
 13. 18. The non-humananimal of claim 16, wherein the mutated Slc30a8 gene comprises thecoding sequence set forth in SEQ ID NO:
 22. 19. The non-human animal ofclaim 1, wherein the non-human animal has decreased mitochondrial geneexpression relative to the non-human animal without the mutation. 20.The non-human animal of claim 1, wherein the non-human animal hasincreased Hvcn1 expression relative to the non-human animal without themutation.
 21. The non-human animal of claim 1, wherein the non-humananimal has normal glucose homeostasis and glucose-induced insulinsecretion on a control chow diet relative to the non-human animalwithout the mutation.
 22. The non-human animal of claim 1, wherein thenon-human animal has a normal metabolic phenotype on a control chow dietrelative to the non-human animal without the mutation.
 23. The non-humananimal of claim 1, wherein the non-human animal has one or more of thefollowing characteristics relative to the non-human animal without themutation: (a) increased glucose-induced insulin secretion when fed thehigh-fat diet; (b) increased pancreatic beta-cell proliferation when fedthe high-fat diet; (c) increased number of pancreatic beta cells whenfed the high-fat diet; and (d) increased fed plasma insulin levels afterblockade of the insulin receptor.
 24. The non-human animal of claim 1,wherein the non-human animal has all of the following characteristicsrelative to the non-human animal without the mutation: (a) increasedglucose-induced insulin secretion when fed the high-fat diet; (b)increased pancreatic beta-cell proliferation when fed the high-fat diet;(c) increased number of pancreatic beta cells when fed the high-fatdiet; and (d) increased fed plasma insulin levels after blockade of theinsulin receptor.
 25. The non-human animal of claim 1, wherein thenon-human animal has one or more of the following characteristicsrelative to the non-human animal without the mutation: (a) increasedcirculating insulin levels after fed the high-fat diet for 20 weeks; (b)increased number of pancreatic beta cells after fed the high-fat dietfor 20 weeks; (c) decreased proinsulin-to-insulin ratio when fed thehigh-fat diet; and (d) increased fed plasma insulin levels afterblockade of the insulin receptor.
 26. The non-human animal of claim 1,wherein the non-human animal has all of the following characteristicsrelative to the non-human animal without the mutation: (a) increasedcirculating insulin levels after fed the high-fat diet for 20 weeks; (b)increased number of pancreatic beta cells after fed the high-fat dietfor 20 weeks; (c) decreased proinsulin-to-insulin ratio when fed thehigh-fat diet; and (d) increased fed plasma insulin levels afterblockade of the insulin receptor.
 27. The non-human animal of claim 1,wherein Slc30a8 mRNA expression levels in the islets of the non-humananimal are at least 25% of Slc30a8 mRNA expression levels in the isletsof the non-human animal without the mutation.
 28. The non-human animalof claim 1, wherein the non-human animal is heterozygous for themutation.
 29. The non-human animal of claim 1, wherein the non-humananimal is homozygous for the mutation.
 30. The non-human animal of claim1, wherein the non-human animal is male.
 31. The non-human animal ofclaim 1, wherein the non-human animal is female.
 32. A method of makingthe non-human animal of claim 1, comprising: (a) contacting the genomeof a non-human animal pluripotent cell that is not a one-cell stageembryo with: (i) an exogenous repair template comprising an insertnucleic acid flanked by a 5′ homology arm that hybridizes to a 5′ targetsequence at the Slc30a8 locus and a 3′ homology arm that hybridizes to a3′ target sequence at the Slc30a8 locus, wherein the insert nucleic acidcomprises the mutation; (ii) a Cas9 protein; and (iii) a guide RNA thathybridizes to a guide RNA recognition sequence within the Slc30a8 locus,wherein the Slc30a8 gene is modified to comprise the mutation; and (b)introducing the modified non-human animal pluripotent cell into a hostembryo; and (c) implanting the host embryo into a surrogate mother toproduce a genetically modified F0 generation non-human animal in whichthe Slc30a8 gene is modified to comprise the mutation, wherein themutation results in the F0 generation non-human animal having anenhanced capacity for insulin secretion relative to a non-human animalwithout the mutation when fed the high-fat diet.
 33. (canceled) 34.(canceled)
 35. A method of making the non-human animal of claim 1,comprising: (a) contacting the genome of a non-human animal one-cellstage embryo with: (i) an exogenous repair template comprising an insertnucleic acid flanked by a 5′ homology arm that hybridizes to a 5′ targetsequence at the Slc30a8 locus and a 3′ homology arm that hybridizes to a3′ target sequence at the Slc30a8 locus, wherein the insert nucleic acidcomprises the mutation; (ii) a Cas9 protein; and (iii) a guide RNA thathybridizes to a guide RNA recognition sequence within the Slc30a8 locus,wherein the Slc30a8 gene is modified to comprise the mutation; and (b)implanting the modified non-human animal one-cell stage embryo into asurrogate mother to produce a genetically modified F0 generationnon-human animal in which the Slc30a8 gene is modified to comprise themutation, wherein the mutation results in the F0 generation non-humananimal having an enhanced capacity for insulin secretion relative to anon-human animal without the mutation when fed the high-fat diet.36.-38. (canceled)
 39. A method of screening a compound for activity forameliorating or exacerbating type-2-diabetes, comprising: (a) contactinga subject non-human animal of claim 1 with the compound; and (b)measuring one or more of the following in the subject non-human animalrelative to a control non-human animal not contacted with the compound,wherein the control non-human animal comprises the same Slc30a8 mutationas the subject non-human animal: (1) glucose-induced insulin secretionwhen fed a high-fat diet; (2) pancreatic beta-cell proliferation levelswhen fed the high-fat diet; (3) number of pancreatic beta cells when fedthe high-fat diet; and (4) fed plasma insulin levels after blockade ofthe insulin receptor, whereby activity for ameliorating type 2 diabetesis identified by one or more of the following in the subject non-humananimal compared with the control non-human animal: (1) increasedglucose-induced insulin secretion when fed the high-fat diet; (2)increased pancreatic beta-cell proliferation when fed the high-fat diet;(3) increased number of pancreatic beta cells when fed the high-fatdiet; and (4) increased fed plasma insulin levels after blockade of theinsulin receptor, and whereby activity for exacerbating type 2 diabetesis identified by one or more of the following in the subject non-humananimal compared with the control non-human animal: (1) decreasedglucose-induced insulin secretion when fed the high-fat diet; (2)decreased pancreatic beta-cell proliferation when fed the high-fat diet;(3) decreased number of pancreatic beta cells when fed the high-fatdiet; and (4) decreased fed plasma insulin levels after blockade of theinsulin receptor.
 40. A method of screening a compound for activity forameliorating or exacerbating type-2-diabetes, comprising: (a) contactinga subject non-human animal of claim 1 with the compound; and (b)measuring one or more of the following in the subject non-human animalrelative to a control non-human animal not contacted with the compound,wherein the control non-human animal comprises the same Slc30a8 mutationas the subject non-human animal: (1) capacity to secrete insulin inresponse to hyperglycemia; (2) insulin clearance; (3) mitochondrial geneexpression; and (4) Hvcn1 expression, whereby activity for amelioratingtype 2 diabetes is identified by one or more of the following in thesubject non-human animal compared with the control non-human animal: (1)increased capacity to secrete insulin in response to hyperglycemia; (2)increased insulin clearance; (3) decreased mitochondrial geneexpression; and (4) increased Hvcn1 expression, and whereby activity forexacerbating type 2 diabetes is identified by one or more of thefollowing in the subject non-human animal compared with the controlnon-human animal: (1) decreased capacity to secrete insulin in responseto hyperglycemia; (2) decreased insulin clearance; (3) increasedmitochondrial gene expression; and (4) decreased Hvcn1 expression.
 41. Anon-human animal cell of the non-human animal of claim 1, wherein thegenome of the non-human animal cell comprises an endogenous Slc30a8locus comprising a mutated Slc30a8 gene, wherein the mutated Slc30a8gene encodes a truncated SLC30A8 protein, and wherein a non-human animalcomprising the mutated Slc30a8 gene has an enhanced capacity for insulinsecretion relative to a non-human animal without the mutation. 42.(canceled)
 43. A targeting vector for generating a mutated Slc30a8 geneat an endogenous Slc30a8 locus in a non-human animal, wherein thetargeting vector comprises a 5′ homology arm targeting a 5′ targetsequence at the endogenous Slc30a8 locus and a 3′ homology arm targetinga 3′ target sequence at the endogenous Slc30a8 locus, wherein thetargeting vector comprises a mutation in the Slc30a8 gene, wherein themutated Slc30a8 gene encodes a truncated SLC30A8 protein, and wherein anon-human animal comprising the mutated Slc30a8 gene has an enhancedcapacity for insulin secretion relative to a non-human animal withoutthe mutation.